The role of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, in regulating the oxidation of fatty acids in rat skeletal (1, 2) and cardiac (3, 4) muscle has been intensively investigated. Recent studies have demonstrated that its concentration in rat muscle is governed, at least in part, by changes in the activity of the muscle isoform of acetyl-CoA carboxylase (ACC ␤ ) 1 (5), the enzyme that catalyzes malonylCoA synthesis. Thus, in keeping with their observed effects on malonyl-CoA concentration and fatty acid oxidation, insulin and glucose appear to activate ACC ␤ in muscle by increasing the cytosolic concentration of citrate, an allosteric activator of ACC ␤ and a precursor of its substrate, cytosolic acetyl-CoA. Conversely, decreases in malonyl-CoA concentration and increases in fatty acid oxidation in muscle during exercise (contraction) have been linked to decreases in ACC ␤ activity, attributable to its phosphorylation and inhibition by the ␣ 2 isoform of AMP-activated protein kinase (AMPK) (5). AMPK can also be activated and the concentration of malonyl-CoA decreased by exposing resting muscle to 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is taken into the muscle and phosphorylated to form the 5Ј-AMP analogue ZMP (6).Whether a change in malonyl-CoA turnover contributes to the alterations in its concentration in muscle during exercise and other conditions is not known. In a lipogenic tissue such as liver, the de novo synthesis of fatty acids is thought to be the major mechanism by which malonyl-CoA is utilized. In contrast, in skeletal muscle fatty acid synthesis occurs at a very low rate, if at all (7), and attention has been focused on malonyl-CoA decarboxylase (MCD) for removal of malonyl-CoA (1). Evidence has been presented that MCD is present in both cardiac (8, 9) and skeletal (1, 10, 11) muscle. In skeletal muscle, its activity is similar to that of ACC (1). In heart, in which MCD activity is substantially greater than in skeletal muscle, a decrease in the K m of MCD for malonyl-CoA has been reported following an increase in its work load (9). On the other hand, no change in activity has been observed following ischemia-reperfusion of the heart, a situation in which AMPK is activated (8). The question of whether MCD is acutely regulated in skeletal muscle and, if so, how has not been studied previously.In this study, we describe the characteristics of purified MCD from rat skeletal muscle and contraction-induced changes in its maximal activity and affinity for malonyl-CoA. In addition, the effects of the AMPK activator 5-aminoimida-