Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca²⁺-dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophin-deficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wild-type cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.