To test whether endothelium-derived nitric oxide (NO) regulates mitochondrial respiration, NO was pharmacologically modulated in the isolated mouse heart, which was perfused at constant flow to sensitively detect small changes in MVO₂. Stimulation of NO formation by 10 µM bradykinin (Bk) increased coronary venous nitrite release 5-fold to 58±33 nM (n=17). Vasodilatation by Bk, adenosine (1µM) or papaverine (10µM) decreased perfusion pressure, LVDP and MVO₂. In presence of adenosine-induced vasodilatation, stimulation of endothelial NO synthesis by Bk had no effect on LVDP and MVO₂. Also inhibition of NO formation by L-NMMA (100 µM) did not significantly alter LVDP and MVO₂. Similarly, intracoronary infusion of authentic NO 2 µM did not influence LVDP or MVO₂ (- 1±1 %). Only when NO was > 2 µM, contractile dysfunction and a reduction in MVO₂ was observed. Since Bk-induced stimulation of endothelial NO formation and basal NO are not sufficient to impair MVO₂ in the saline perfused mouse heart, a tonic control of the respiratory chain by endothelial NO is difficult to conceive.