DNA replication is initiated upon binding of “initiators” to origins of replication. In simian virus 40 (SV40), the core origin contains four pentanucleotide binding sites organized as pairs of inverted repeats. Here we describe the crystal structures of the origin binding domain (obd) of the SV40 large T-antigen (T-ag) both with and without a subfragment of origin-containing DNA. In the co-structure, two T-ag obds are oriented in a head-to-head fashion on the same face of the DNA, and each T-ag obd engages the major groove. Although the obds are very close to each other when bound to this DNA target, they do not contact one another. These data provide a high-resolution structural model that explains site-specific binding to the origin and suggests how these interactions help direct the oligomerization events that culminate in assembly of the helicase-active dodecameric complex of T-ag., Author Summary How DNA replicates is a critical question for understanding life. DNA replication remains difficult to investigate in eukaryotes, where it involves a complex, multi-protein apparatus which initiates replication at multiple poorly-defined DNA sequences. This process is far easier to study in viral systems, where the DNA sequences at the origin of replication are well-defined and only one or two proteins are required to initiate replication. In simian virus 40 (SV40), the large T-antigen protein (T-ag) is responsible for recognizing DNA sequences required to start replication, called the origin of replication. SV40 T-ag can also cause DNA to melt or unwind. We report here the crystal structure of the DNA-binding domain of SV40 T-ag on a DNA fragment derived from the viral origin of replication. The structure shows that although T-ag and its functionally analogous protein, papilloma virus E1, share no detectable sequence homology in this region, the two domains bind the DNA in similar ways. In both cases, DNA binding is thought to initiate assembly of a complex of the full-length proteins on DNA. Interestingly, SV40 T-ag DNA-binding domains do not interact with one another when bound to DNA. In addition to describing the molecular details of the DNA–protein interactions and the alterations in protein structure induced by DNA binding, we present a model describing the subsequent assembly events., Crystal structures of the SV40 large T antigen (T-ag) origin binding domain (obd) with and without DNA show how two T-ag obds orient head-to-head and engage with the major groove.