Objective: The role of nitric oxide (NO) in myocardial ischemia/reperfusion injury remains controversial as both NO donors and NO synthase (NOS) inhibitors have shown to be protective. We generated transgenic (TG) mice that overexpress endothelial NOS (eNOS) exclusively in cardiac myocytes to determine the effects of high cardiac NO levels on ischemia/reperfusion injury and cellular Ca²⁺ homeostasis. Wild-type (WT) mice served as controls. Methods: Hearts were perfused in vitro and subjected to 20 min of total no-flow ischemia and 30 min of reperfusion (n=5 per group). Left ventricular function, cGMP levels and intracellular Ca²⁺ transients (Ca²⁺_i) were determined. Results: Left ventricular pressure was reduced (maximum, −33%) and basal cardiac cGMP was increased (twofold) in TG hearts, and the changes were reversed by NOS blockade with N^G-nitro-l-arginine methyl ester (l-NAME). Relative to baseline, recovery of reperfusion contractile function was significantly better in hearts from TG (98%) than WT (51%) mice, and l-NAME abolished this effect. Heart rate and coronary perfusion pressure were not different between groups. Systolic and diastolic Ca²⁺_i concentrations were similar in WT and TG hearts, but Ca²⁺_i overload during early reperfusion tended to be less in TG hearts. Kinetic analysis of pressure curves and Ca²⁺_i transients revealed a faster left ventricular diastolic relaxation and abbreviated aequorin light signals in TG hearts at baseline and during reperfusion. Conclusions: High levels of NO/cGMP strongly protect against ischemia/reperfusion injury, the protection is largely independent of changes in Ca²⁺_i modulation, but relates to reduced preischemic performance. Myocyte-specific NO augmentation may aid in studies of the (patho)physiological roles of cardiac-derived NO.