The present experiment determined the effects of glutathione and ascorbic acid, the two most important hydrophilic antioxidants, on myocardial ischemia-reperfusion injury and evaluated their relative therapeutic values. Isolated rat hearts were subjected to ischemia (30 min) and reperfusion (120 min) and treated with ascorbic acid, glutathione monoethyl ester (GSHme), or their combination at the onset of reperfusion. Administration of 1 mM GSHme alone, but not 1 mM ascorbic acid alone, significantly attenuated postischemic injury (P < 0.05 versus vehicle). Most interestingly, coadministration of ascorbic acid with GSHme markedly enhanced the protective effects of GSHme (P < 0.01 versus vehicle). The protection exerted by the combination of GSHme and ascorbic acid at 1 mM each was significantly greater than that observed with 1 mM GSHme alone (P < 0.05). Moreover, treatment with GSHme alone or GSHme plus ascorbic acid markedly reduced myocardial nitrotyrosine levels, suggesting that these treatments attenuated myocardial peroxynitrite formation. These results demonstrated that 1) GSHme, but not ascorbic acid, exerted protective effects against ischemia-reperfusion injury; and 2) the protective effects of GSHme were further enhanced by coadministration with ascorbic acid, suggesting a synergistic effect between GSHme and ascorbic acid.