Measurement of pyruvate and lactate produced from glucose by confluent skin fibroblast cultures from 95 patients with lactic acidemia revealed 10 in whom the lactate/pyruvate ratio (L/P) was increased (L/P = 57 to 232) compared with that observed in control cell lines (L/P = 18 to 35). Mitochondria prepared from these cells revealed two types of respiratory chain defect. In four patients the deficient activity was present in NADH-coenzyme Q reductase (14% to 21% of controls), and in six the deficiency was in cytochrome c oxidase (21% to 28% of controls). The four patients with NADH-coQ reductase deficiency presented early with lactic acidosis, respiratory failure, anorexia, and hypotonia; all four died within 7 months. The group with cytochrome oxidase deficiency had a somewhat later (18 months to 2 years of age) presentation with milder lactic acidemia, but also with hypotonia and anorexia. They had delayed development, beginning to walk and talk at 18 to 24 months, and then slowly regressed. Although an investigation of central nervous system disorders in this latter group has not been possible, the clinical progression fits into the broad category of Leigh disease. We conclude that in these two groups respiratory chain defects can be detected and localized by the use of skin fibroblast cultures.