Evidence to identify the cellular sources of oxy-radical generation in myocardium has been of an indirect nature. We have used low-temperature ESR spectroscopy to identify and characterize ischemia-induced changes in myocardial paramagnetic metabolites. Iron-sulfur proteins associated with the NADH or succinate dehydrogenases of the mitochondrial electron-transport chain were progressively reduced with the onset and development of ischemia. This study provides direct evidence for ischemia-induced changes in an intracellular source of superoxide radical generation that may contribute to oxy-radical production during reperfusion.