Prostaglandin E2 (PGE2) and prostaglandin F(2α) (PGF(2α)) inactivated glycogen synthase and activated glycogen phosphorylase in rat hepatocytes in a dose- and time-dependent manner. These effects were dependent on the presence of Ca2+ in the incubation medium. When glycogen synthase was immunoprecipitated from cells incubated with [32P]P(i) and then treated with PGE2 or PGF(2α), there was increased phosphorylation of the 88 kDa subunit of the enzyme. This phosphorylation affected two CNBr fragments of the glycogen synthase, CB-1 and CB-2, the same fragments that are phosphorylated by different glycogenolytic hormones. No phosphorylation of glycogen synthase by prostaglandins was observed in the absence of Ca2+. Thus the effect of PGE2 and PGF(2α) on these glycogen-metabolizing enzymes supports a role for regulation by prostaglandins of glucose metabolism in parenchymal liver cells.