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. Furthermore, the role of free radicals in the development of reperfusion arrhythmias and functional disturbances also was investigated using a 30-min period of global ischemia followed by 30 min of reperfusion in the isolated working rat heart. The spin trapping agent 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) when it perfused the heart, 100 mumoles/liter, during the first 10 min of reperfusion attenuated the development of reperfusion arrhythmias and improved the functional recovery of the heart during reperfusion. Without treatment, 55% of hearts showed irreversible ventricular fibrillation, and this was completely prevented by DMPO. In DMPO-treated hearts, the recovery of heart function was improved; thus, coronary flow, aortic flow, left ventricular developed pressure, and first derivative of left ventricular developed pressure were significantly increased from their maximal control values of 16.2 +/- 1.9 ml/min, 12.7 +/- 0.9 ml/min, 11.1 +/- 0.5 kPa, and 426 +/- 31 kPa/s to 21.8 +/- 1.3 ml/min (p less than 0.05), 28.4 +/- 3.0 ml/min (p less than 0.001), 14.5 +/- 1.0 kPa (p less than 0.01), and 584 +/- 41 kPa/s (p less than 0.01), respectively. Left ventricular end-diastolic pressure was also significantly reduced from its control value of 2.8 +/- 0.2 kPa to 2.1 +/- 0.2 kPa (p less than 0.05), while the recovery of heart rate was not improved by DMPO treatment. Parallel ESR studies using DMPO as spin trap demonstrated the formation of .OH radicals in the effluent of the reperfused hearts. ESR signals of the formed DMPO-OH, alpha N = alpha beta H = 1.48 mT, were observed within the first seconds of reperfusion with peak concentrations after about 3 min. In the first series of ESR studies, DMPO (200 mmol/liter) was mixed up effluent and ESR signals were recorded, while in the second series of studies, DMPO was directly infused into the heart. Both methods were appropriate to demonstrate the radical formation that peaked at 3 min of reperfusion after 30 min of global ischemia. Cardiotoxic effects of DMPO can be excluded by using of the "mix-up" method (DMPO is added to effluent) because relatively high DMPO concentration (20-200 mmol/liter) is important for demonstration of free radical production.(ABSTRACT TRUNCATED AT 400 WORDS)