The eukaryotic initiation factor 2 (eIF-2) from calf brain has been purified to homogeneity and free of endogenous kinase activity. Phosphorylation of eIF-2 factor has been examined with four different protein kinases. Casein kinase II, calcium/phospholipid-dependent protein kinase and cyclic AMP-dependent protein kinase from brain, phosphorylate the beta subunit of eIF-2, whilst hemin-controlled inhibitor phosphorylate the alpha subunit of the factor. According to the peptide maps obtained, the phosphorylation sites of the factor by the three beta kinases are specific and distinct. These data suggest a different regulation for the beta subunit through this modification.