Malonyl-CoA decarboxylase is the main route for the disposal of malonyl-CoA, the key metabolite in the regulation of mitochondrial fatty acid oxidation. We have developed a simple and sensitive radiochemical assay to determine malonyl-CoA decarboxylase activity. The decarboxylation of [2-14C]malonyl-CoA produces [2-14C]acetyl-CoA, which is converted to [2-14C]acetylcarnitine in the presence of excess L-carnitine and carnitine acetyltransferase. The positively charged radiolabeled product, acetylcarnitine, is separated from negatively charged excess radiolabeled substrate and the radioactivity measured by scintillation counting. Measurement of malonyl-CoA decarboxylase activities with this method gives values comparable to those obtained with assays currently in use, but has the advantage of being simpler and less labor intensive. We have applied this assay to rat skeletal muscle of different fiber-type composition and to rat heart. Malonyl-CoA decarboxylase activity (mU/g wet wt) correlates with the oxidative capacity of the muscles, being lowest in type IIb fibers (42.7 +/- 3.0) and highest in heart (1071.4 +/- 260), with intermediate activity in type IIa fibers (150.7 +/- 4.3) and type I fibers (107.8 +/- 7.6). Studies on subcellular distribution of malonyl-CoA decarboxylase activity in rat heart and rat skeletal muscle show that approximately 50 and 65% is localized to mitochondria, while 50 and 35% of the activity is extramitochondrial.