Previously, it has been shown that *NO donor S-nitrosoglutathione (GSNO) improves the postischemic functional recovery in crystalloid buffer-perfused isolated rat hearts subjected to cardioplegic ischemia. Supplementation of cardioplegic solution with nitronyl nitroxide, a scavenger of *NO, antagonized this protective effect. Using low temperature ESR, we have detected nitrosylmyoglobin (MbNO) in rat hearts subjected to cardioplegic ischemia in the presence of GSNO (20-200 mumol/l). During aerobic reperfusion MbNO signal intensity gradually decreased, but persisted for up to 30 min of aerobic reperfusion. We conclude that MbNO is an endogenous marker of *NO release in myocardial tissues. Implications of MbNO formation are discussed with respect to cardioprotection during ischemia- and reperfusion-induced myocardial injury.