Endogenous nitric oxide (eNO) modulates tissue respiration. To test whether eNO modulates myocardial O2consumption (MV˙o2), ATP synthesis, and metabolic efficiency, we used isolated isovolumic guinea pig hearts perfused at a constant flow. Nω-nitro-l-arginine (l-NNA; 5 × 10−5mol/l) was used to inhibit eNO production. MV˙o2was measured at different levels of cardiac work, estimated as the rate-pressure product (RPP). ATP content and synthesis rate were determined using31P NMR and magnetization transfer during high cardiac work. l-NNA increased coronary vascular resistance (19 ± 3%, P < 0.05) and MV˙o2(12 ± 3%, P< 0.05) without an increase in the RPP. In contrast, vehicle infusion resulted in insignificant changes in coronary vascular resistance (3 ± 2%, P > 0.05) and MV˙o2(−2 ± 1%, P> 0.05). Compared with vehicle, l-NNA caused a higher MV˙o2both during KCl arrest (l-NNA 5.6 ± 0.5 vs. vehicle 3.0 ± 0.4 μmol · min−1· mg dry wt−1, P < 0.05) and during increased cardiac work elicited by elevating perfusate Ca2+, indicating an upward shift in the relationship between contractile performance (measured as RPP) and MV˙o2. However, neither ATP contents nor ATP synthesis rates were different in the two groups during high cardiac work. Thus, because inhibition of eNO production byl-NNA increased MV˙o2without a change in the ATP synthesis rate, these data suggest that eNO increases myocardial metabolic efficiency by reducing MV˙o2in the heart.