Generation of superoxide radicals by the mitochondrial respiratory chain of cardiomyocites isolated from rat heart and treated with saponin was studied. The rate of O2- production was measured by electron paramagnetic resonance (EPR) spectroscopy using hydroxylamine TEMPONE-H as spin trap. A device has been constructed which provided permanent stirring of cardiomyocyte samples directly in the cavity and prevented cell aggregation. When substrates and antimycin A and/or rotenone are added, the radical production rate increased and reached its maximum in the presence of the both inhibitors. Superoxide dismutase as well as KCN suppressed the radical production, thus being suggestive of the generation of superoxide radicals in the bc1 complex, while the mechanism of O2- production is the same as was suggested for isolated mitochondria. The ratio between rates of O2- generation by isolated cardiomyocytes under various experimental conditions is in a good accord with corresponding parameter of isolated mitochondria. However, in the case of cardiomyocytes the absolute values of the O2- production rate are approximately twice as high as those in isolated mitochondria, presumably due to the partial damage of the mitochondrial respiratory chain during the isolation procedure.