Activities for the oxidation of palmitoylcarnitine, of palmitoyl-CoA and of carnitine palmitoyltransferase were measured in mitochondria prepared from needle biopsy samples of human skeletal muscle. Results are presented for nine normal subjects and 18 patients in whom there was evidence of mitochondrial abnormality. Palmitoylcarnitine and palmitoyl-CoA oxidation were measured spectrophotometrically by following the reduction of added cytochrome c in the presence of cyanide. Because of large variations in the activities between subjects it was essential to express the three activities per unit of cytochrome c oxidase activity to demonstrate unambiguous specific alterations in the activities. In most of the patients the order of the three activities was similar to that in the normal subjects. However, in five cases the activity for palmitoylcarnitine oxidation was less than 4% of the mean normal value. In two of these patients, the low activity could be accounted for by very low (less than 10% normal) activity of carnitine palmitoyltransferase (CPT). In another two patients the activity of CPT was normal but that of palmitoyl-CoA dehydrogenase (a measure of beta-oxidation) was very low.