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A high molecular weight rabbit reticulocyte protein factor Co-eIF-2 regulates eIF-2 activity during initiation of protein synthesis. Co-eIF-2 contains Co-eIF-2A and Co-eIF-2C activities. Co-eIF-2A stimulates Met-tRNA(,f) binding to eIF-2 and stabilizes the ternary complex. Co-eIF-2C stimulates Met-tRNA(,f) binding to eIF-2 in the presence of Mg('2+) by relieving Mg('2+) inhibition of ternary complex formation from eIF-2. Co-eIF-2 protein complex contains several polypeptides including M(,r) 80,000 and 50,000 polypeptides. Three polypeptides (M(,r) 80,000, 50,000 and 25,000) are present in 0.5M KCl ribosomal salt wash and each possesses Co-eIF-2A activity. M(,r) 80,000 polypeptide (Co-eIF-2A('80)) has been purified to homogeneity and its properties studied: (1) Co-eIF-2A('80) stimulated Met-tRNA(,f) binding to eIF-2 and the complex formed was resistant to aurintricarboxylic acid. (2) Co-eIF-2A('80) activity was NEM resistant and heat-labile. (3) Antibodies prepared against homogenous Co-eIF-2A('80) strongly inhibited protein synthesis in reticulocyte lysates and, also, eIF-2 and Co-eIF-2 promoted Met-tRNA(,f) binding to 40S ribosomes. Inhibition of protein synthesis in reticulocyte lysates was overcome by preincubation of anti-Co-eIF-2A('80) with homogenous Co-eIF-2A('80) and was partially overcome by similar preincubation with Co-eIF-2. (4) Upon limited digestion with Staphylococcus aureus V8 protease, the homogenous Co-eIF-2A('80) gave two major polypeptide fragments (M(,r) 50,000 and 25,000). Upon similar treatment, an M(,r) 80,000 polypeptide band isolated from the SDS-gel of the Co-eIF-2 complex gave four major polypeptide fragments, and two of these fragments (M(,r) 50,000 and 25,000) were similar to those given by Co-eIF-2A('8-), indicating that this M(,r) 80,000 polypeptide band contains the Co-eIF-2A('80) component. In another study, we isolated the messenger RNA corresponding to Co-eIF-2A('80) from erythroleukemia cells. The procedure was: (1) Screening of