Troglitazone (CS-045) is a new type of antidiabetic agent that decreases plasma glucose by enhancing insulin action in insulin-resistant diabetic animals and non-insulin-dependent diabetes mellitus (NIDDM) patients. To examine the direct effect of troglitazone on glucose metabolism and insulin action in skeletal muscle, we infused troglitazone solution into perfused rat hindlimbs in the presence of 6 mmol/L glucose and in the absence or presence of insulin. In the absence of insulin, even 50 mumol/L troglitazone did not elicit glucose uptake. Troglitazone did increase lactate and pyruvate release at concentrations of 20 mumol/L and higher; however, it decreased the ratio of lactate to pyruvate (L/P ratio) and increased oxygen consumption at concentrations higher than 5 and 20 mumol/L, respectively. In hindlimb muscle, 20 mumol/L troglitazone decreased glycogen content without changing fructose 2,6-bisphosphate (F2,6P2) content in the absence of insulin. Insulin infusion with 250 microU/mL obtained half-maximal effects, causing a 2.8-fold increase in glucose uptake and a 1.5-fold increase in lactate and pyruvate release. When 20 mumol/L troglitazone was infused for 30 minutes together with 250 microU/mL insulin, insulin-induced glucose uptake significantly increased 30 minutes after troglitazone infusion, and this increase was further augmented after withdrawal of troglitazone. In insulin plus troglitazone infusion at 30 minutes after troglitazone removal, glycogen content in hindlimb muscle was significantly decreased compared with that obtained with insulin infusion alone. In summary, in the absence of insulin, troglitazone does not elicit glucose uptake, but causes an increase in glycolysis accompanied by a decrease in muscle glycogen content and L/P ratio and an increase in oxygen consumption. In the presence of insulin, troglitazone increases insulin-induced glucose uptake, and this increase is further augmented after troglitazone removal. Addition of troglitazone to insulin infusion decreased the glycogen content in hindlimb muscle. This decrease in muscle glycogen content may trigger an enhancement of insulin-induced glucose uptake similar to that observed during muscle contraction or epinephrine treatment.