Yeast hexokinase 2 is known to be a phosphoprotein in vivo, prominently labeled from 32P-inorganic phosphate after a shift of cells to medium with low glucose concentration [Vojtek, A. B., & Fraenkel D. G. (1990) Eur. J. Biochem, 190, 371-375]. The principal and perhaps sole site of phosphorylation is now identified as residue serine-15, by observation of a single tryptic peptide difference, its sequencing and size determination by mass spectrometry, and by mutation to alanine, which prevents phosphorylation in vivo. Although protein kinase A was unlikely to accomplish the phosphorylation in vivo, serine-15 does belong to a protein kinase A consensus phosphorylation sequence, and in vitro phosphorylation by protein kinase A at serine-15 could be shown by labeling and by peptide determination. The alanine-15 mutant enzyme was not phosphorylated in vitro.