Nitric oxide (NO) may limit myocardial ischemia-reperfusion injury by slowing mitochondrial metabolism. We examined whether rat heart contains catalysts potentially capable of reducing nitrite to NO during an episode of regional myocardial ischemia produced by temporary coronary artery occlusion. In intact Sprague-Dawley rats, a 15-minute left coronary occlusion lowered the nitrite concentration of myocardial regions exhibiting ischemic glucose metabolism to ~50% that of non-ischemic regions (185±223 vs. 420±203 nmol L^-1). Nitrite was rapidly repleted during subsequent reperfusion. Heart tissue tested in vitro acquired a substantial ability to consume nitrite when made hypoxic at neutral pH, and this ability was slightly enhanced by simultaneously lowering pH to 5.5. More than 70% of this activity could be abolished by flushing the coronary circulation with crystalloid to remove trapped erythrocytes. Correspondingly, erythrocytes demonstrated the ability to reduce exogenous nitrite to NO under hypoxic conditions in vitro. In erythrocyte-free heart tissue, nitrite consumption increased 5-fold when pH was lowered to 5.5 . Approximately 40% of this pH-sensitive increase in nitrite consumption could be blocked by the xanthine oxidoreductase (XOR) inhibitor allopurinol, whereas lowering pO2 sufficiently to desaturate myoglobin accelerated it further. We conclude that rat heart contains several factors capable of catalyzing ischemic nitrite reduction; the most potent is contained within erythrocytes and activated by hypoxia, while the remainder include XOR and other pH-sensitive factors endogenous to heart tissue, including deoxymyoglobin.