Preincubation of rat hepatocytes with EGF (epidermal growth factor) caused a stimulation of gluconeogenesis from alanine. The effect was maximal after preincubation of 20 min, and a half-maximal effect of EGF was obtained at 10 nM. EGF also stimulated gluconeogenesis from lactate and asparagine, but not from glutamine or from proline. Preincubation of hepatocytes with EGF caused a stable inactivation of pyruvate kinase, which may account, at least in part, for the observed effects of EGF on gluconeogenesis.