Leigh disease associated with cytochrome c oxidase deficiency (LD^[COX−]) is one of the most common disorders of the mitochondrial respiratory chain, in infancy and childhood. No mutations in any of the genes encoding the COX-protein subunits have been identified in LD^(COX−) patients. Using complementation assays based on the fusion of LD^(COX−) cell lines with several rodent/human rho⁰ hybrids, we demonstrated that the COX phenotype was rescued by the presence of a normal human chromosome 9. Linkage analysis restricted the disease locus to the subtelomeric region of chromosome 9q, within the 7-cM interval between markers D9S1847 and D9S1826. Candidate genes within this region include SURF-1, the yeast homologue (SHY-1) of which encodes a mitochondrial protein necessary for the maintenance of COX activity and respiration. Sequence analysis of SURF-1 revealed mutations in numerous DNA samples from LD^(COX−) patients, indicating that this gene is responsible for the major complementation group in this important mitochondrial disorder.