Incorporation of 32P into yeast fructose-1,6-bisphosphatase (EC 3.1.3.11) was observed after addition of glucose to a cell suspension incubated with (32P)orthophosphoric acid. The 32P counts were coincident with the enzyme band when immunoprecipitates were subjected to sodium dodecyl sulfate disc gel electrophoresis. The incorporation of phosphate was associated with a decrease in enzyme activity. Approximately 1 mol of phosphate was incorporated/mol of enzyme. The phosphate is bound to the enzyme in a phosphoester linkage with a serine residue. Release of 32P accompanying enzyme reactivation was observed both in vivo and in cell-free extracts.