Cultured skin fibroblasts from four patients with Leigh syndrome and cytochrome c oxidase deficiency were studied. Mitochondrial DNA (mtDNA) analysis excluded large-scale deletions and known point mutations associated with Leigh syndrome. The COX activities were reduced to 18-44% of healthy probands, when measured in the presence of laurylmaltoside. COX activity from patients was shown to be more temperature sensitive than COX activity from control cells. In order to determine the subunit composition of COX immunoblotting studies were performed using mono- and polyclonal antibodies to distinct subunits. A monoclonal antibody to subunit IV crossreacted with two unknown proteins of higher apparent molecular weight in mitochondria from three patients, but not in mitochondria from control and the fourth patient. Quantification of immunoreactivity revealed a decrease of subunits II/III and IV parallel to the determined enzyme activity. In contrast, a variable amount of subunit VIIa (and/or VIIb) was found in mitochondria from different patients. The results indicate a defective COX holoenzyme complex in patients with Leigh syndrome and suggest different molecular origins of the defect.