The contribution of endogenous NO to ischemia-reperfusion injury was studied in isolated perfused hearts of wild-type (WT) and endothelial NO synthase knockout (eNOS⁻) mice. The hearts were subjected to a 16-min period of global no-flow ischemia and were subsequently reperfused for 1 h. Cardiac contractile function was evaluated and³¹P-NMR spectroscopy was used to monitor myocardial energy status and the intracellular pH. During both baseline and ischemia, there were neither significant differences in mechanical function nor in energetic parameters between the two groups, for example at baseline left ventricular developed pressure (LVDP) was 56.5±5.4 mmHg in WT and 58.7±5.2 mmHg in eNOS⁻and phosphocreatine (PCr) level was 12.9±1.3 mmin WT and 12.7±1.7 mmin eNOS⁻. In reperfusion, however, a significant improvement of the post-ischemic functional and metabolic recovery became apparent in the eNOS⁻hearts. While in the WT group, LVDP recovered only to 38.4±5.3 mmHg, LVDP in the eNOS⁻group attained 49.4±5.5 mmHg at the end of 60 min reperfusion (P<0.05,n=8). Similarly, the recovery of PCr was significantly enhanced in the transgenic hearts as compared to WT (10.4±1.6vs8.1±1.3 mm,P<0.05). eNOS⁻hearts also showed a better restoration of dP/dtand a significant lower left ventricular enddiastolic pressure. In an additional series of wild-type hearts, the NO synthase inhibitor N^G-monomethyl-l-arginine methyl ester (100μm) also tended to improve the recovery of both LVDP (43.8±6.8 mmHg) and PCr (9.5±1.6 mm) in reperfusion (1 h), but the restoration of functional and metabolic parameters was less pronounced when compared with eNOS⁻. The results provide clear evidence that endogenously formed NO significantly contributes to ischemia-reperfusion injury in the saline-perfused mouse heart, most likely by peroxynitrite formation from NO.