Nitric oxide (NO) is a ubiquitous signaling molecule that can inhibit cytochrome c oxidase (COX), the terminal complex of the mitochondrial respiratory chain. Some bacteria possess an NAD(P)H-dependent NO dioxygenase that degrades NO to nitrate, thereby protecting against NO toxicity. There is evidence for the existence of a similar mechanism in plant and animal cells, although the subcellular location of the enzymes involved is unknown. In this work, we investigated the occurrence of this activity in mitochondria by studying the effect of NAD(P)H on the inhibition of respiratory activity by S-nitrosoglutathione (GSNO), an NO donor. The oxygen consumption by mitochondria isolated from potato tubers and rat liver was measured with a Clark-type electrode and the mitochondrial membrane potential was estimated spectrophotometrically using safranine. The oxygen consumption by NAD(P)H-energized potato tuber mitochondria was only transiently inhibited by GSNO. In contrast, GSNO persistently inhibited oxygen consumption when mitochondria were energized with succinate or N-N-N'-N'-tetramethyl-p-phenylenediamine (TMPD). This inhibition was reverted in the presence of NAD(P)H, even in situations in which the oxidation of NAD(P)H  by the respiratory chain was prevented. This effect was enhanced when potato mitochondria were ruptured. At concentrations that caused collapse of the mitochondrial membrane potential produced by succinate oxidation, GSNO caused only a transient fall in the potential generated by NAD(P)H in potato tuber mitochondria. In experiments with succinate-energized rat liver mitochondria, NAD(P)H also reverted the inhibition of the respiratory activity caused by GSNO. In contrast to potato mitochondria, this effect was not seen in uncoupler-treated or ruptured rat liver mitochondria, indicating an important role of the mitochondrial membrane potential in this process in animal mitochondria. Overall, these results indicate that NAD(P)H accelerates NO degradation, decreasing the inhibitory effect of this radical on COX. Based on these findings, we suggest the existence of a mitochondrial NAD(P)H-dependent mechanism for NO metabolism, possibly an NO dioxygenase, in potato tubers and rat liver. This mechanism may protect the mitochondrial respiratory chain against the deleterious effects of NO.