The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate partially neutralized the stimulatory effects of epinephrine (alpha 1-adrenergic actions), glucagon, and dibutyryl-cAMP on gluconeogenesis in isolated hepatocytes of fasted rats, when lactate or dihydroxyacetone was used as the substrate. By constructing metabolic crossover plots and by comparing rates of lactate production from dihydroxyacetone with K0.5 values of extracted pyruvate kinase for phosphoenolpyruvate, we obtained evidence that phorbol ester actions on hormonally stimulated gluconeogenesis were accompanied by proportionate increases in activity of pyruvate kinase. Although purified pyruvate kinase from rat liver was a substrate for protein kinase C in vitro, phosphorylation was not accompanied by modulation of kinetic parameters. Furthermore, incubation of pyruvate kinase extracted from hormone-treated hepatocytes with protein kinase C revealed no activation of the prephosphorylated enzyme. This and the absence of effects of the phorbol ester on basal rates of gluconeogenesis and lactate production suggest that effects of protein kinase C on pyruvate kinase activity in hepatocytes may result from impairment of steps at the level of hormone-induced signal transduction.