Alterations in the production of nitric oxide (NO.) are a critical factor in the injury that occurs in ischemic and reperfused myocardium; however, controversy remains regarding the alterations in NO. that occur and how these alterations cause tissue injury. As superoxide generation occurs during the early period of reperfusion, the cytotoxic oxidant peroxynitrite (ONOO-) could be formed; however, questions remain regarding ONOO- formation and its role in postischemic injury. Electron paramagnetic resonance spin trapping studies, using the NO. trap Fe(2+)-N-methyl-D-glucamine dithiocarbamate (Fe-MGD), and chemiluminescence studies, using the enhancer luminol, have been performed to measure the magnitude and time course of NO. and ONOO- formation in the normal and postischemic heart. Isolated rat hearts were subjected to control perfusion, or ischemia followed by reperfusion in the presence of Fe-MGD with electron paramagnetic resonance measurements performed on the effluent from these hearts. Whereas only trace signals were present prior to ischemia, prominent NO. adduct signals were seen during the first 2 min of reflow. The reperfusion associated increase in these NO. signals was abolished by nitric oxide synthase inhibition. In hearts perfused with luminol to detect ONOO- formation, a similar marked increase was seen during the first 2 min of reperfusion that was blocked by nitric oxide synthase inhibitors and by superoxide dismutase. Either NG-nitro-L-arginine methyl ester or superoxide dismutase treatment resulted in more than twofold higher recovery of contractile function than in untreated hearts. Immunohistology studies demonstrated that the ONOO(-)-mediated nitration product nitrotyrosine was formed in postischemic hearts, but not in normally perfused controls. Thus, NO. formation is increased during the early period of reperfusion and reacts with superoxide to form ONOO-, which results in protein nitration and myocardial injury.