Although cytochrome c oxidase (COX) deficiency is recognised to present from the neonatal period to adult life, in the majority of cases its molecular genetic basis has not been defined. We have identified a 36 year old woman with recurrent encephalopathy associated with lactic acidosis, proximal myopathy and exercise induced myalgia. Muscle histochemistry showed that 90% of the fibres exhibited very low or absent COX activity and there were no ragged red fibres. Enzymatic studies identified an 80% reduction of COX activity and cytochrome studies revealed a marked reduction in cytochrome aa3 at 23% of normal. Immunocytochemistry with antibodies to both mitochondrial and nuclear encoded subunits revealed a pattern suggestive of a primary mitochondrial DNA (mtDNA) defect in the COX deficient fibres and consistent with either reduced stability or impaired assembly of the holoenzyme. Sequence analysis of mtDNA identified a heteroplasmic G to A point mutation at position 9952 in skeletal muscle which was not detectable in leukocyte mtDNA and not identified in 120 healthy controls or 50 patients with other mitochondrial encephalomyopathies. Mass myoblast and fibroblast cultures did not harbour the mutation. This mutation creates a premature stop codon in the highly conserved C terminal region of the COX III subunit which predicts the loss of the last 12 amino acids. Single fibre polymerase chain reaction studies provided further evidence for an association between this mutation and COX deficiency. This is the first stop codon mutation identified in human mitochondrial DNA.