Glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), in isolated rat hepatocytes, has been identified as a novel intracellular target for tumor-promoting phorbol esters such as phorbol 12-tetradecanoate 13-acetate (TPA). Exposure of hepatocytes to TPA resulted in a 50% decrease in the activity ratio of glycogen synthase without/with glucose 6-phosphate. The inactivation was dose dependent and was half-maximal at a TPA concentration of approximately 16 nM (10 ng/ml). Phorbol and phorbol 13-monoacetate, ineffective tumor promoters, had little influence on glycogen synthase activity. Other biologically active diesters, phorbol 12,13-didecanoate, phorbol 12,13-dibutyrate, and phorbol 12,13-dibenzoate, caused significant inactivation of glycogen synthase. Glycogen phosphorylase (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) activity, however, was unaffected by TPA or any of the tumor-promoting phorbol esters mentioned above. It is concluded that phorbol diesters can interact in the regulatory pathway for glycogen synthase, but the lack of effect on phosphorylase argues that distinct mechanisms can operate for the control of glycogen synthase and phosphorylase.