This study was undertaken to estimate the extent of molecular defects in the mitochondrial electron-transfer chain of a patient with mitochondrial myopathy. Biochemical and immunochemical studies were performed on the skeletal muscle mitochondria. Spectrophotometry and enzyme activity measurements localized a definite defect at the segment of cytochromecoxidase (complex IV) of the electron-transfer chain. Immunoblotting and immunoprecipitation studies using the anti-complex IV antibody revealed that the contents of subunits 1, 4, 5, 6, and 7 of complex IV were markedly diminished and that subunit 2 was almost absent. Inummohistochemistry of the muscle tissue revealed a considerable accumulation of immunoreactive materials of complex IV in the ragged-red fibers. The immunoblots using the anti-NADH-ubiquinone oxidoreductase antibody demonstrated that the contents of NADH-ubiquinone oxidoreductase subunits were 47% of control and that the contents of three subunits were considerably decreased. The contents of ubiquinol-cytochromecoxidoreductase subunits were also somewhat low (77% of control) and one of the minor contaminants detected in the control was completely absent. High-resolution one-dimensional sodium dodecyl sulfate-urea-gel electrophoresis disclosed that six additional unidentified polypeptides in the control were markedly diminished or completely missing. These results demonstrate that the molecular defects in the mitochondrial electron-transfer chain are more extensive than would be expected from either spectral analysis or enzyme activity measurements alone, and involve not only complex IV but also NADH-ubiquinone oxidoreductase and ubiquinol-cytochromecoxidoreductase and several unidentified mitochondrial proteins. (Pediatr Res24: 447–454, 1988)