The skeletal muscle of a patient with a mitochondrial myopathy was examined. A defect in the electron transport chain was identified at the position of complex III by activity measurements and the low levels of reducible cytochrome b. The polypeptide composition of complex III in the patient's mitochondria was determined by antibody binding experiments. The method allowed detection of individual polypeptides at a lower limit of 10-40 ng of protein. Characterization of protein composition is thus possible by using a biopsy sample of 1 g of tissue. The level of core proteins, FeS protein, and subunit VI was greatly diminished in the patient's mitochondria. Cytochrome c1 polypeptide was found at normal levels but was sensitive to proteolysis by trypsin. These results show that complex III is not assembled in the patient's mitochondria. The possible role of cytochrome b as the site of the primary lesion is discussed.