The effect of alanine on pyruvate kinase was investigated in isolated rat hepatocytes. Alanine at concentrations of 2, 5 or 10 mM increased glucose production by 73% during the first 30 minutes of incubation of hepatocytes with 9 mM lactate and 1 mM pyruvate. After 3 minutes, the rate was not affected by the addition of alanine. A dose-response study showed that maximal stimulation of gluconeogenesis was achieved with 0.5 mM alanine. Using a method that measured recycling of phosphoenolpyruvate to pyruvate, it was found that in either fed or starved rats, alanine significantly decreased the percentage of phosphoenolpyruvate recycling when lactate was the substrate. However, no significant change in recycling was noted when either pyruvate or lactate-pyruvate was the glucose precursor. This study suggests that in intact liver cells, alanine has an inhibitory effect on pyruvate kinase. However, the inhibition is not of sufficient magnitude to completely account for the increase in glucose production when lactate is the substrate. It is hypothesized that alanine may have other effects on gluconeogenesis in addition to that of inhibiting pyruvate kinase.