Mitochondria are affected by endogenous nitric oxide (NO). Besides effects of NO on mitochondrial enzymes and the stimulation of mitochondrial H2O2 production, a NO-dependent increase in mitochondrial biogenesis in several tissues has been reported. It is still obscure whether NO generated by one specific or different NO synthase (NOS) isoenzymes determine such effects. Therefore, we analyzed the amount of mitochondria, respiratory chain enzyme complexes, and citrate synthase in the brain, muscle, heart, kidney, and liver by comparing wild-type (WT) mice and mice lacking the neuronal nitric oxide synthase isoform (nNOS-KO). Our results show that the activities of NADH:cytochrome c oxidoreductase and succinate cytochrome c oxidoreductase differ between WT and nNOS-KO mice. However, similar quantities of mitochondria were found in the homogenates of tissues in WT and nNOS-KO animals. Most impressive, higher activities and protein of citrate synthase were found in the brain, muscle, heart, kidney, and liver of nNOS-KO mice. Additionally, higher contents of fatty acid synthase and lipids were determined in the livers of nNOS-KO mice but not in the heart and brain. Furthermore, liver mitochondria from nNOS-KO mice consumed pyruvate at a higher rate and released more citric acid. Our data document a previously unrecognized role of endogenous NO in the regulation of lipid metabolism.