We show by the use of 32P-labeling in vivo that hexokinase 2 and hexokinase 1 in Saccharomyces cerevisiae are phosphoproteins. The highest labeling was after incubation in medium with a low concentration of glucose, when labeling appears to be predominant even without use of immunoprecipitation. The nature of the modification is not known, but it has properties consistent with a phosphomonoester of serine or threonine. The cAMP-dependent protein kinase plays a negative role in hexokinase phosphorylation, in that there was reduced labeling in strains (bcyl) lacking a regulatory subunit, and increased labeling during growth with high concentrations of glucose in a strain attenuated in the catalytic subunit (tpklw1). The function of the modification is not known, but there was a correlation between the extent of labeling and the expression of kinase-dependent high-affinity glucose uptake.