The ability of iron chelates to promote hydroxyl radical (.OH) formation from hydrogen peroxide (H2O2) via Fenton chemistry was exploited to detect H2O2 produced during the oxidations of the eumelanin precursors 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA). H2O2 generation during the autooxidations of DHI and DHICA was confirmed on the basis of the electrochemical detection of three hydroxylation products of salicylate [2,3 and 2,5-dihydroxybenzoic acid (DHBA) and catechol], which was used as an .OH indicator. The oxidations of both 5,6-dihydroxyindoles were augmented by tyrosinase and peroxidase without the addition of H2O2. The partial inhibitions by catalase of the auto-oxidations and tyrosinase- and peroxidase-mediated oxidations of DHI and DHICA provide additional evidence of an endogenous origin of H2O2 during the final stages of eumelanogenesis. The mechanism proposed for the formation of H2O2 involves the semiquinones of DHI and DHICA in the univalent transfer of electrons to molecular oxygen. The observations described in this study support previous reports suggesting that factors modulating the levels of H2O2 in melanocytes and melanoma cells play critical roles in directing the course of melanogenesis and influencing the potential cytotoxicity of the biosynthetic pathways.