The role of substrate availability in the regulation of gluconeogenesis in isolated rat hepatocytes was studied using [U-14C]alanine as a tracer in the presence of different concentrations of L-alanine in the incubation medium. At low alanine concentrations (0.5 mM) insulin decreased the 14C incorporation into the glucose pool and increased the incorporation of tracer carbons into the protein and lipid pools and into CO2. The net radioactivity lost from the glucose pool was only a small percentage of the total increase in the activity of the protein, lipid, CO2, or glycogen pools, supporting the notion that the effect of insulin in diminishing gluconeogenesis is secondary to its effects on pathways using pyruvate. At higher concentrations of alanine (2.5, 5.0, and 10.0 mM) in the incubation medium insulin increased the movement of alanine carbons into protein and glucose. This suggests that at higher substrate concentrations the ability of the liver to synthesize proteins is overwhelmed and the pyruvate carbons are forced into the gluconeogenesis pathway. These results were further confirmed by using [U-14C]lactate. The increases in observed specific activity of glucose following insulin administration would not be possible if insulin acted by affecting the activity of any enzyme directly involved in the formation or utilization of pyruvate, most of which have been proposed as sites of insulin action. Data presented show that insulin "inhibits" gluconeogenesis by affecting a change in substrate availability.