The indoleamine melatonin is ubiquitously distributed, and because of its small size and amphiphilic nature, it is able to reach easily all cellular compartments. The highest intracellular melatonin concentrations are found in the mitochondria, suggestive of local metabolism and/or direct participation in organelle function. In mitochondria cytochrome c (cyt c) could represent a melatonin target since it has the capability to oxidize organic molecules in the presence of H2O2, and mitochondria are the main site of H2O2 production in nonphagocytic cells. Therefore, we investigated oxidation of melatonin by cyt c/H2O2 couple as a potential pathway for its metabolism in the mitochondria. We found melatonin conversion into N1-acetyl-N2-formyl-5- methoxykynuramine via sequential steps that generate the intermediates 2-hydroxymelatonin and 2,3-dihydroxymelatonin. We experimentally excluded mediation by a Fenton/Haber-Weiss-type reaction and documented the dependence on oxoferryl heme for melatonin oxidation. Given the high mitochondrial concentrations of both melatonin and cyt c as well as the continuous generation of H2O2 during respiration, it is entirely possible that mitochondrial cyt c-mediated oxidation of melatonin may be a plausible pathway of its biotransformation in vivo. © 2005 American Chemical Society.