Nitric oxide (NO*) plays an important role in various physiological processes. The aim of the present study was to investigate if brain mitochondrial nitric oxide synthase (mtNOS) is active and functional during hypertension. L-citrulline production, an indicator of nitric oxide synthesis, was concentration-dependent on L-arginine in all strains and all ages tested, and was inhibited by 7-Nitroindazole (7-NI). Brain mitochondria of 1 month-old (prehypertensive) spontaneously hypertensive rats (SHR) exhibited a significantly (p < 0.05) low basal L-citrulline content as compared to age-matched Wistar (W) and Wistar-Kyoto (WKY) rats. L-citrulline synthesis in SHR rats showed a significant (p < 0.01) low response to L-arginine in 3 and 7 months-old rats. Respiratory rates in states 3 and 4 increased with low L-arginine concentration in all strains and all ages. The results suggest that in rat brain mitochondria, L-citrulline synthesis is constant once age-related hypertension is installed and NO* does not regulate oxidative phosphorylation.