The purpose of this study was to use a direct method, that of electron spin resonance (ESR) spectroscopy, to demonstrate that reperfusion after a period of ischemia results in a sudden increase in the production of free radicals in the myocardium. The isolated buffer-perfused rat heart was used with N-tert-butyl-alpha-phenylnitrone (PBN) as a spin-trapping agent. Samples of coronary effluent were taken and extracted into toluene for detection of radical adducts by ESR spectroscopy. After 15 minutes of total, global ischemia, aerobic reperfusion resulted in a sudden burst of radical formation that peaked at 4 minutes. When hearts were reperfused with anoxic buffer, no dramatic increase in radical production was observed. Subsequent reintroduction of oxygen, however, resulted in an immediate burst of radical production of a similar magnitude to that seen in the wholly aerobic reperfusion experiments. The ESR signals obtained (aN = 13.60 G, aH = 1.56 G) are consistent with the spin-trapping by PBN of either a carbon-centered species or an alkoxyl radical, both of which could be formed by secondary reactions of initially-formed oxygen radicals with membrane lipid components.