Malonyl-CoA, the inhibitor of carnitine acyltransferase I, is an important regulator of fatty acid oxidation and ketogenesis in the liver. Muscle carnitine acyltransferase I has previously been reported to be more sensitive to malonyl-CoA inhibition than is liver carnitine acyltransferase I. Fluctuations in malonyl-CoA concentration may therefore be important in regulating the rate of fatty acid oxidation in muscle during exercise. Male rats were anesthetized (pentobarbital via venous catheters) at rest or after 30 min of treadmill exercise (21 m/min, 15% grade). The gastrocnemius/plantaris muscles were frozen at liquid N2 temperature. Muscle malonyl-CoA decreased from 1.66 +/- 0.17 to 0.60 +/- 0.05 nmol/g during the exercise. This change was accompanied by a 31% increase in cAMP in the muscle. The decline in malonyl-CoA occurred before muscle glycogen depletion and before onset of hypoglycemia. Plasma catecholamines, corticosterone, and free fatty acids were all significantly increased during the exercise. This exercise-induced decrease in malonyl-CoA may be important for allowing the increase in muscle fatty acid oxidation during exercise.