The site specificity of phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) by the heme-regulated and double-stranded RNA-activated eIF-2alpha kinases were compared by phosphopeptide mapping. eIF-2alpha was maximally phosphorylated in vitro with [gamma-(32)P]ATP and either crude or partially purified preparations of the kinases. (32)P-Labeled eIF-2alpha was isolated by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. The fixed, stained, and dried polypeptide band was excised and then exhaustively digested directly in the gel slice with one of several proteases (trypsin, chymotrypsin, subtilisin, or thermolysin); the resultant [(32)P]phosphopeptides were analyzed by one-dimensional chromatography or by two-dimensional chromatography and high-voltage electrophoresis. In addition, limited proteolysis of [(32)P]eIF-2alpha contained in fixed, dried, and stained gel slices was achieved with Staphylococcus aureus protease V8, chymotrypsin, or subtilisin, and the partial (32)P-labeled cleavage products were analyzed by gel electrophoresis. Each protease produced distinct and reproducible [(32)P]phosphopeptide profiles after partial or exhaustive proteolysis of [(32)P]eIF-2alpha. With a given protease, identical [(32)P]phosphopeptide patterns were obtained whether eIF-2alpha was phosphorylated by the heme-regulated or the double-stranded RNA-activated kinase. These data indicate that, in vitro, the kinases phosphorylate sites on eIF-2alpha that are identical or proximally located in the primary sequence. In this report we also provide preliminary evidence that the two eIF-2alpha kinases activated in lysates by heme deficiency or double-stranded RNA phosphorylate site(s) of endogenous eIF-2alpha that are similar, if not identical, to the sites phosphorylated in vitro with partially purified eIF-2alpha kinase(s) and eIF-2.