Muscle and skin biopsy were taken from subjects 1–3 and referred to local centers for metabolic investigations; additionally, array comparative genomic hybridization was performed for subject 3 but revealed no abnormalities. Respiratory-chain analyses of muscle biopsy confirmed isolated complex I deficiency in subjects 1 and 2 (44% and 46% of control subjects, respectively), whereas analysis of muscle from subject 3 revealed normal complex I enzymology despite clinically suggestive features of mitochondrial disease. Diagnostic studies of respiratory-chain complex assembly (2D Blue Native polyacrylamide gel electrophoresis [BN-PAGE]) were undertaken in fibroblasts from subject 3 and revealed a marked complex I assembly defect in isolation, supporting a clinical diagnosis of complex I deficiency. Informed consent for diagnostic and research studies was obtained for all subjects in accordance with the Declaration of Helsinki protocols and approved by local institutional review boards.