Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.