Respiratory parameters of cardiac mitochondria were determined in the bundles of cardiac fibers skinned by using saponin that specifically removed sarcolemma, but left intracellular structures intact. In the assay medium which simulated the ion composition of cardiac cytoplasm maximal value of state 3 oxygen consumption per mol cytochromes aa₃ was close to that value for isolated mitochondria. Ischemia and isopreterenol treatment were found to affect respiratory parameters of mitochondria in saponin-skinned fibers, among them creatine-stimulated respiration decreased most significantly, (3–4)-times under these conditions. The method described can be easily applied for determination of the mitochondrial respiratory parameters in small (5–10 mg) biopsy samples from human heart.