Melatonin prevents mitochondrial failure in models of sepsis through its ability to inhibit the expression and activity of both cytosolic (iNOS) and mitochondrial (i-mtNOS) inducible nitric oxide synthases. Because Parkinson's disease (PD), like sepsis, is associated with iNOS induction, we assessed the existence of changes in iNOS/i-mtNOS and their relation with mitochondrial dysfunction in the MPTP model of PD, which also displays increased iNOS expression. We also evaluated the role of melatonin (aMT) and its brain metabolite, N1-acetyl-5-methoxykynuramine (AMK), in preventing i-mtNOS induction and mitochondrial failure in this model of PD. Mitochondria from substantia nigra (SN) and, to a lesser extent, from striatum (ST) showed a significant increase in i-mtNOS activity, nitrite levels, oxidative stress, and complex I inhibition after MPTP treatment. MPTP-induced i-mtNOS was probably related to mitochondrial failure, because its prevention by aMT and AMK reduced oxidative/nitrosative stress and restored complex I activity. These findings represent the first experimental evidence of a potential role for i-mtNOS in the mitochondrial failure of PD and support a novel mechanism in the neuroprotective effects of aMT and AMK. © 2009 Wiley-Liss, Inc.