The renal and cardiac benefits of renin-angiotensin system (RAS) inhibition in hypertension exceed those attributable to blood pressure reduction, and seem to involve mitochondrial function changes. To investigate whether mitochondrial changes associated with RAS inhibition are related to changes in NO metabolism, 4 groups of male Wistar rats were treated during 2 weeks with a RAS inhibitor, enalapril (10 mg /kg.d, Enal), or a NOS inhibitor, L-NAME (N-Nitro-L-Arginine Methyl Ester) (1 mg /kg.d,), or both (Enal+L-NAME), or were untreated (Control). Blood pressure and body weight were lower in Enal than in Control. Electron transfer through complexes I to III and cytochrome oxidase activity were significantly lower, and uncoupling protein-2 content significantly higher, in kidney mitochondria isolated from Enal than in those from Control. All these changes were prevented by L-NAME co-treatment, and were accompanied by a higher production/bioavailability of kidney NO. L-NAME abolished mitochondrial NOS activity, but failed to inhibit extra-mitochondrial kidney NOS, underscoring the relevance of mitochondrial NO in those effects of enalapril that were suppressed by L-NAME co-treatment. In Enal, kidney mitochondria H₂O₂ production rate and MnSOD activity were significantly lower than in Control, and these effects were not prevented by L-NAME co-treatment. These findings may clarify the role of NO in the interactions between RAS and mitochondrial metabolism, and can help unravel the mechanisms involved in renal protection by RAS inhibitors.