During ischemia and reperfusion, with an increase in intracellular Na+ and a depolarized membrane potential, Ca2+ may enter the myocyte in exchange for intracellular Na+ via reverse-mode Na+-Ca2+ exchange (NCX). To test the role of Ca2+ entry via NCX during ischemia and reperfusion, we studied mice with cardiac-specific ablation of NCX (NCX-KO) and demonstrated that reverse-mode Ca2+ influx is absent in the NCX-KO myocytes. Langendorff perfused hearts were subjected to 20 minutes of global ischemia followed by 2 hours of reperfusion, during which time we monitored high-energy phosphates using 31P-NMR and left-ventricular developed pressure. In another group of hearts, we monitored intracellular Na+ using 23Na-NMR. Consistent with Ca2+ entry via NCX during ischemia, we found that hearts lacking NCX exhibited less of a decline in ATP during ischemia, delayed ischemic contracture, and reduced maximum contracture. Furthermore, on reperfusion following ischemia, NCX-KO hearts had much less necrosis, better recovery of left-ventricular developed pressure, improved phosphocreatine recovery, and reduced Na+ overload. The improved recovery of function following ischemia in NCX-KO hearts was not attributable to the reduced preischemic contractility in NCX-KO hearts, because when the preischemic workload was matched by treatment with isoproterenol, NCX-KO hearts still exhibited improved postischemic function compared with wild-type hearts. Thus, NCX-KO hearts were significantly protected against ischemia-reperfusion injury, suggesting that Ca2+ entry via reverse-mode NCX is a major cause of ischemia/reperfusion injury.