—We tested the hypothesis that nitric oxide (NO) released by endothelial NO synthase (eNOS) is not only important in blood pressure regulation but also involved in cardiac function and remodeling and in the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEi). With the use of a 2D Doppler echocardiography system equipped with a 15-MHz linear transducer, we evaluated left ventricular (LV) morphology and function in conscious eNOS knockout mice (eNOS^−/−; n=15) and their wild-type littermates (eNOS^+/+; n=16). We also studied whether in eNOS^−/− mice (1) myocardial ischemia/reperfusion injury is more severe and (2) the cardioprotective effect of ACEi is diminished or absent. In comparison with the wild type, eNOS^−/− mice had significantly increased systolic blood pressure (128±3 versus 108±5 mm Hg; P<0.001) and decreased heart rate (531±22 versus 629±18 bpm; P<0.001) associated with increased LV posterior wall thickness (0.80±0.04 versus 0.64±0.02 mm; P<0.001) and LV mass (18.3±0.9 versus 13.1±0.5 mg/10 g body weight; P<0.01). Despite hypertension and LV hypertrophy, LV chamber dimension, shortening fraction and ejection fraction (indicators of LV contractility), and cardiac output did not differ between the 2 strains, which indicates that LV function in eNOS^−/− mice is well compensated. We also found that in eNOS^+/+ mice, ACEi decreased the ratio of myocardial infarct size to area at risk from 62.7±3.9% to 36.3±1.6% (P<0.001), whereas in eNOS^−/− mice this effect of ACEi was almost abolished: the ratio of myocardial infarct size to area at risk was 67.2±2.9% in the vehicle-treated group and 62.7±3.9% in mice treated with ACEi. Moreover, infarct size in vehicle-treated eNOS^−/− mice was not significantly different from eNOS^+/+ mice given the same treatment. We concluded that (1) endothelium-derived NO plays an important role in the regulation of blood pressure homeostasis; (2) NO released under basal conditions has no significant impact on cardiac function; and (3) ACEi protect the heart against ischemia/reperfusion injury in mice and that this effect is mediated in part by endothelium-derived NO.