While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation of that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alteration in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurements of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia.