The present study demonstrates that mitochondrial cytochrome c reacts with the thiol-reacting agent N-ethylmaleimide (NEM) to produce a one NEM-adducted cytochrome c. Mitochondrial cytochrome c also reacts with 5,5'-dithio-bis-(2-nitrobenzoic acid) and 1-chloro-2,4-dinitrobenzene in a manner prevented with NEM or iodoacetic acid (IAA). NEM-treated cytochrome c has lower reducibility and lower function to support mitochondrial oxygen consumption. These findings suggest that mitochondrial cytochrome c contains a reactive thiol that is involved in the functions of cytochrome c for mitochondria. Nitric oxide reacts with the cytochrome c thiol to generate S-nitroso (SNO)-cytochrome c in a manner prevented with NEM or IAA. SNO-cytochrome c has lower reducibility and function to support mitochondrial oxygen consumption, similar to NEM-treated cytochrome c.