Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S.mRNA. eIF3.Met-tRNAf.eIF2.GTP) and mediates hydrolysis of the bound GTP. To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude initiation factor preparations. We observed that eIF5 specifically interacted with the beta subunit of initiation factor eIF2. No other initiation factors including the gamma subunit of eIF2 tested positive in this assay. Furthermore, both yeast and mammalian eIF5 bind to the beta subunit of either mammalian or yeast eIF2. Binding analysis with human eIF2beta deletion mutants expressed in Escherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2beta. These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNAf.eIF2.GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the beta subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation.