Cytochrome c release from mitochondria is essential for apoptosis. Using human myelogenous leukemia ML-1a, its respiration-deficient and reconstituted cells, we demonstrated that respiratory function is essential for tumor necrosis factorinduced cytochrome c release. In a cell free system using mitochondrial fraction from ML-1a, initiation of respiration by substrates for complexes I, II, and III but not IV released cytochrome c, suggesting that reduction of coenzyme Q or complex III is essential for cytochrome c release. In the same system, disruption of mitochondrial outer membrane was neither enough nor the cause for cytochrome c release by succinate. These observations define an early pathway in which a change in respiration releases cytochrome c. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.