Initiation factor 4E (eIF-4E) binds to the m7GTP-containing cap of eukaryotic mRNA and facilitates the entry of mRNA into the initiation cycle of protein synthesis. eIF-4E is a phosphoprotein, and the phosphorylated form binds to mRNA caps 3-4-fold more tightly than the nonphosphorylated form. A previous study indicated that the major phosphorylation site was Ser-53 (Rychlik, W., Russ, M. A., and Rhoads, R. E. (1987) J. Biol. Chem. 262, 10434-10437). In the present study, we synthesized the phosphopeptide expected to result from tryptic digestion of eIF-4E, O-phosphoseryllysine. Surprisingly, the tryptic and synthetic phosphopeptides did not comigrate electrophoretically. Accordingly, we redetermined the phosphorylation site by isolating a chymotryptic phosphopeptide on reverse phase high performance liquid chromatography. The peptide was sequenced by Edman degradation and corresponded to 198QSHADTATKSGSTTKNRF215. The site of phosphorylation was determined to be Ser-209 by four methods: the increase in the ratio of dehydroalanine to serine derivatives during Edman degradation, the release of 32P, the further digestion of the chymotryptic phosphopeptide with trypsin, Glu-C, and Asp-N, and site-directed mutagenesis of eIF-4E cDNA. The S209A variant was not phosphorylated in a rabbit reticulocyte lysate system, whereas the wild-type, S53A, and S207A variants were. This site falls within the consensus sequence for phosphorylation by protein kinase C.