Mitochondrial Ca2+ accumulation and the formation of reactive oxygen species are processes dependent on the electron transport system. The production of superoxide by respiring rat heart mitochondria was decreased by either chelating extramitochondrial Ca2+ with EGTA or by blocking mitochondrial Ca2+ uptake with ruthenium red. Mitochondrial experiments with doxorubicin showed an enhanced stimulation of reactive oxygen species, which was also inhibited by EGTA or ruthenium red. Myocardial cell cultures treated with doxorubicin showed an enhanced formation of intracellular reactive oxygen species, which preceded cell damage. Ruthenium red not only attenuated the enhanced formation of intracellular reactive oxygen species, but also increased cell viability. The relationship between mitochondrial Ca2+ transport and the formation of superoxide suggests that a disruption in mitochondrial Ca2+ homeostasis by doxorubicin may be involved in the release of reactive oxygen species and its cardiotoxicity.