Telomeres are guanine-rich, simple repeat sequences that constitute the physical ends of eukaryotic chromosomes. The sequence and structure of these specialized DNA termini permits extension of the G strand by the enzyme telomerase, thereby counteracting sequence loss due to incomplete replication or degradative activities (Lingner et al. 1995; Greider 1996). In yeast and human cells, a telomerase deficiency leads to a progressive decline in telomere length that inhibits the proliferative capacity of the cell and heralds replicative senescence (Lundblad and Szostak 1989; Bodnar et al. 1998). Analysis of telomerase-deficient mice has also revealed an essential role for telomere maintenance in the long-term viability of high-renewal organ systems (Lee et al. 1998). Furthermore, loss of telomerase in the absence of checkpoint function can also promote genetic instabilities and carcinogenesis, especially in highly regenerative tissues (Chin et al. 1999; Artandi et al. 2000), illustrating the importance of telomeres in maintaining genomic stability. Increasing attention has been directed at the importance of coordinating G-strand synthesis by telomerase with replication of the C strand of the telomere. Following extension of the 3′ terminus of the G-rich strand by telomerase, fill-in synthesis of the other strand, presumably by the machinery that normally performs lagging strand DNA replication, is thought to be necessary to prevent elongated single-stranded regions at chromosome termini. Several recent studies indicate that disruption of C-strand DNA synthesis can impair telomere length control (for review, see Price 1997; Evans and Lundblad 2000). In the ciliate Euplotes, inhibition of DNA polymerases α and δ during de novo telomere synthesis alters the length of the telomeric C strand and also causes an increase in the length and heterogeneity of the G strand (Fan and Price 1997). Similarly, propagation of S. cerevisiae strains defective for components of the lagging strand DNA replication machinery cause a telomerase-dependent increase in telomere length (Carson and Hartwell 1985; Adams and Holm 1996; Adams Martin et al. 2000). In addition, de novo telomere formation at a newly created double-strand break requires not only telomerase but also DNA polymerase α and δ (Diede and Gottschling 1999). Collectively, these results indicate that in both yeast and ciliates, G-strand and C-strand synthesis are tightly coregulated, although the molecular mechanism for this coordination has not been elucidated. In yeast, a critical contributor to several aspects of telomere function is the protein Cdc13, which exhibits high affinity sequence-specific binding for single-stranded telomeric DNA (Lin and Zakian 1996; Nugent et al. 1996; Hughes et al. 2000a). Consistent with this substrate specificity, Cdc13 is associated with the telomere during S phase (C.I. Nugent and V. Lundblad, in prep.), which correlates with the transient increased single-strand G-rich extension observed at yeast telomeres (Wellinger et al. 1993, 1996). CDC13 performs an essential activity necessary to protect chromosome termini, as loss of CDC13 function is accompanied by immediate and extensive loss of one strand of the telomere (Garvik et al. 1995; Diede and Gottschling 1999). In addition to this end protection function, CDC13 positively regulates telomere replication by recruiting telomerase to the telomere, an activity that is abolished by the cdc13-2 missense mutation. The cdc13-2 strain, which is normal for telomeric end protection, exhibits a telomere replication defect similar to that of a strain that lacks telomerase (Nugent et al. 1996), although telomerase activity in cell-free cdc13-2 extracts is unaffected (Lingner et al. 1997). This defect is partially suppressed by increased expression of the telomerase-associated Est1 protein (Nugent et al. 1996), and a complex containing both proteins can be coimmunoprecipitated in yeast, using overexpressed recombinant versions of Est1 and Cdc13 (Qi and Zakian 2000). Furthermore, the cdc13-2 mutation can be reciprocally suppressed by a specific missense mutation in EST1 (Pennock et al. 2001), thereby demonstrating that positive regulation of telomere replication by Cdc13 is due to a direct interaction between Cdc13 and a subunit of telomerase, resulting in recruitment of the enzyme to the telomere. In addition to this positive regulatory role, Cdc13 has been implicated in negative control of telomere length. The thermolabile cdc13-1 mutant exhibits greatly elongated telomeres when grown at intermediate temperatures (Grandin et al. 1997). However, this mutant strain is also severely impaired for the essential end protection function of CDC13 (Garvik et al. 1995; Diede and Gottschling 1999); therefore, analysis of the cdc13-1 mutation has not clarified whether the proposed role in negative length control is distinct from the essential function of CDC13. An additional factor that contributes to negative length regulation is the Cdc13-interacting protein Stn1. These two proteins associate in a two-hybrid assay and display a number of genetic interactions, suggesting that Stn1 and Cdc13 function together at the telomere as a complex (Grandin et al. 1997). Furthermore, a mutation in STN1 increases telomere length by ∼1 kb, leading Charbonneau and colleagues to propose that Stn1 is a negative regulator of telomere length (Grandin et al. 1997, 2000). Cdc13 is also associated with DNA polymerase α, although mutations that disrupt this association have only a modest (50–150 bp) increase in telomere length (Qi and Zakian 2000); therefore, the contribution of the Cdc13-Pol α association to telomere length control is unclear. We describe here the identification of a mutation of CDC13, cdc13-5, that exhibits a striking defect in negative regulation of telomere length, with no impact on the essential function of CDC13. In this strain, telomere length is increased by ≥1000 bp as a result of unregulated elongation of the G-rich strand by telomerase. This is accompanied by a reduced ability to coordinate synthesis of the C-rich strand, resulting in chromosome termini with greatly extended single-strand extensions of the G strand. The cdc13-5 defect is dependent on the ability to recruit telomerase to the telomere, as cdc13-5-mediated telomere elongation is blocked by the cdc13-2 mutation. Genetic interactions between the cdc13-5 mutation and DNA polymerase α also implicate misregulation of C-strand synthesis in this mutant phenotype. Therefore, cdc13-5 defines a role for Cdc13 in coordination of the synthesis of the two strands of the telomere, which is distinct from the telomerase recruitment activity defined by the cdc13-2 mutation. We propose that these two regulatory activities of Cdc13 correspond to two distinct steps in telomere replication that coordinate and regulate synthesis of the two strands of the telomere. In the first step, Cdc13 initiates telomere replication by recruiting telomerase, which extends the 3′ terminus of the G strand of the telomere. In the second step, fill-in synthesis of the C strand by lagging strand synthesis machinery acts to limit extension of the G strand by telomerase, an inhibitory activity that is defective in the cdc13-5 mutant. We further suggest that these two steps are the consequence of successive binding of the telomerase-associated Est1 subunit and the negative regulator Stn1 to overlapping binding sites on Cdc13. By relying on a single protein as the regulatory center, this proposed mechanism provides a simple means for coordinating synthesis of the two strands of the telomere.