In isolated perfused livers of 24-h fasted rats, perfused with lactate (2 mM), pyruvate (0.5 mM), or dihydroxyacetone (1 mM), infusion of tolbutamide (0.5 mM) very rapidly (within 3 min) inhibited the rate of gluconeogenesis. However, gluconeogenesis from fructose (1 mM) and glycerol (1 mM) was not affected by tolbutamide. Tolbutamide also inhibited by 30% the rate of 14CO2 production from livers perfused with [1-14C]pyruvate, without altering the rate of 14CO2 production from [2-14C]pyruvate. The rate of hepatic glycolysis from fructose, glycerol, and dihydroxyacetone was also stimulated by 250, 40, and 100%, respectively, during tolbutamide infusion into perfused livers. Tolbutamide also inhibited the endogenous rate of hepatic ketogenesis by 30%. All of the tolbutamide-mediated alterations in hepatic metabolism were reversed upon withdrawal of tolbutamide from the perfusion medium. Decreased hepatic gluconeogenesis from lactate and pyruvate in the presence of tolbutamide was not a consequence of increased pyruvate oxidation via the pyruvate dehydrogenase complex or the tricarboxylic acid cycle.