Nitric oxide (NO) is produced in mammals by different isoforms of NO synthase (NOS), including the constitutive mitochondrial enzyme (mtNOS). Here we demonstrate that the concentration of NO resulting from a mitochondrial NOS activity increases under hypoxic conditions in isolated rat liver mitochondria. We show that mitochondrially derived NO mediates the impairment of active (state 3) respiration as measured in the presence of the substrates glutamate and malate after reoxygenation. Simultaneously, NO induces oxidative stress in mitochondria, characterized by an increase in the amount of protein carbonyls and a decrease in glutathione (GSH). Both the accumulation of oxidative stress markers during and the impaired respiration after reoxygenation were prevented by blocking NO production with the NOS inhibitor L-NAME. These observations suggest that mitochondria are exposed to high amounts of NO generated by a mitochondrial NOS upon hypoxia/reoxygenation. Such increased NO levels, in turn, inhibit mitochondrial respiration and may cause oxidative stress that leads to irreversible impairment of mitochondria.