To investigate the effect of nitric oxide (NO) on cardiac energy metabolism, isolated cardiomyocytes of Wistar rats were incubated in an Oxystat system at a constant ambient PO₂ (25 mmHg) and oxygen consumption (O₂); free intracellular Ca²⁺ (fura 2), free cytosolic adenosine [S-adenosylhomocysteine (SAH) method], and mitochondrial NADH (autofluorescence) were measured after application of the NO donor morpholinosydnonimine (SIN-1). In Na⁺-free medium (contracting cardiomyocytes), O₂ increased from 7.9 ± 1.2 to 26.4 ± 3.1 nmol · min¹ · mg protein¹. SIN-1 (100 µmol/l) decreased O₂ in contracting (21 ± 3%) and in quiescent cells (24 ± 7%) by the same extent. Inhibition of O₂ was dose dependent (EC₅₀: 10⁷ mol/l). S-nitroso-N-acetyl-penicillamine, another NO donor, also inhibited O₂, whereas SIN-1C (100 µmol/l), the degradation product of SIN-1, displayed no inhibitory effect. Intracellular Ca²⁺ remained unchanged, and inhibition of protein kinases G, A, or C did not antagonize the effect of NO. Mitochondrial NADH increased with NO, indicating a reduced flux through the respiratory chain. In quiescent but not in contracting cardiomyocytes, NO significantly increased adenosine, indicating a reduced energy status. These data suggest the following. 1) NO decreases cardiac respiration, most likely via direct inhibition of the respiratory chain. 2) Whereas in quiescent cardiomyocytes the inhibition of aerobic ATP formation by NO causes reduction in energy status, contracting cells are able to compensate for the NO-induced inhibition of oxidative phosphorylation, maintaining energy status constant.