To test whether endothelium-derived nitric oxide (NO) regulates mitochondrial respiration, NO was pharmacologically modulated in isolated mouse hearts, which were perfused at constant flow to sensitively detect small changes in myocardial O2 consumption (MV̇O2). Stimulation of NO formation by 10 μM bradykinin (BK) increased coronary venous nitrite release fivefold to 58 ± 33 nM ( n = 17). Vasodilatation by BK, adenosine (1 μM), or papaverine (10 μM) decreased perfusion pressure, left ventricular developed pressure (LVDP), and MV̇O2. In the presence of adenosine-induced vasodilatation, stimulation of endothelial NO synthesis by BK had no effect on LVDP and MV̇O2. Also, inhibition of NO formation by NG-monomethyl-l-arginine (l-NMMA, 100 μM) did not significantly alter LVDP and MV̇O2. Similarly, intracoronary infusion of authentic NO ≤2 μM did not influence LVDP or MV̇O2 (-1 ± 1%). Only when NO was >2 μM were contractile dysfunction and MV̇O2 reduction observed. Because BK-induced stimulation of endothelial NO formation and basal NO are not sufficient to impair MV̇O2 in the saline-perfused mouse heart, a tonic control of the respiratory chain by endothelial NO is difficult to conceive.