Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.