Incorporation of 32P from [gamma-32P]ATP into a homogenous preparation of rat hepatic fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphatase 1-phosphohydrolase, EC 3.1.3.11) was catalyzed by a homogeneous preparation of the catalytic subunit of cyclic AMP-dependent protein kinase from bovine liver. Approximately 4 mol of phosphate were incorporated per mol of the tetrameric enzyme. This phosphorylation was associated with an increase in enzyme activity. In addition, in vivo phosphorylation of the enzyme was observed after injection of radioactive inorganic phosphate into rats and subsequent isolation of the enzyme by conventional purification methods and by immunoprecipitation. All of the labeled phosphate incorporation into the enzyme, both in vitro and in vivo, was precipitated by antibody specific for the enzyme. Furthermore, the 32Pi counts were coincident with the enzyme subunit band when the immunoprecipitates were examined by sodium dodecyl sulfate/disc gel electrophoresis. Acid hydrolysis of the immunoprecipitated enzyme that was phosphorylated in vitro revealed that only seryl residues were labeled. On the basis of the concentration of protein kinase (0.2-1.0 muM) necessary to phosphorylate physiological amounts of fructose-1,6-bisphosphatase (1.0-4.0 muM), it is suggested that cyclic AMP-dependent protein kinase may catalyze the phosphorylation of fructose-1,6-bisphosphatase in vivo.