The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) isolated from reticulocyte lysate protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinases. We have now studied the roles of p67 and eIF-2 kinases in regulation of protein synthesis using several animal cell lysates and an animal cell line (KRC-7) in culture under various growth conditions. The results are as follows. (i) Both p67 and eIF-2 kinase(s) are present in active forms in all animal cells under normal growth conditions and p67 protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation, thus promoting protein synthesis in the presence of active eIF-2 kinases. (ii) In heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture, p67 is deglycosylated and subsequently degraded. This leads to eIF-2 kinase-catalyzed eIF-2 alpha-subunit phosphorylation and thus to protein synthesis inhibition. (iii) Addition of a mitogen (namely, phorbol 12-myristate 13-acetate) to serum-starved KRC-7 cells in culture induces an increase of p67 and thus increases protein synthesis. These results suggest the following conclusions. (i) Protein synthesis inhibition in a heme-deficient reticulocyte lysate is not due to the activation of an eIF-2 kinase (heme-regulated inhibitor), as is generally believed, but is due to degradation of p67. The heme-regulated inhibitor is present in an active form and possibly in equal amounts in both heme-deficient and heme-supplemented reticulocyte lysates but cannot phosphorylate eIF-2 alpha subunit because of the presence of p67. (ii) p67 is essential for protein synthesis as it protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of one or more active eIF-2 kinases present in all animal cells. (iii) p67 is both degradable and inducible. Only the p67 level correlates directly with the protein synthesis activity of the cell, indicating that p67 is a critical factor in protein synthesis regulation in animal cells.