A potential new metabolic pathway of melatonin biotransformation is described in this investigation. Melatonin was found to directly scavenge hydrogen peroxide (H 2 O 2 ) to form N 1 -acetyl-N 2 -formyl-5-methoxykynuramine and, thereafter this compound could be enzymatically converted to N 1 -acetyl-5-methoxykynuramine by catalase. The structures of these kynuramines were identified using proton nuclear magnetic resonance, carbon nuclear magnetic resonance, and mass spectrometry. This is the first report to reveal a possible physiological association between melatonin, H 2 O 2 , catalase, and kynuramines. Melatonin scavenges H 2 O 2 in a concentration-dependent manner. This reaction appears to exhibit two distinguishable phases. In the rapid reaction phase, the interaction between melatonin and H 2 O 2 reaches equilibrium rapidly (within 5 s). The rate constant for this phase was calculated to be 2.3 x 10 6 M - 1 s - 1 . Thereafter, the relative equilibrium of melatonin and H 2 O 2 was sustained for roughly 1 h, at which time the content of H 2 O 2 decreased gradually over a several hour period, identified as the slow reaction phase. These observations suggest that melatonin, a ubiquitously distributed small nonenzymatic molecule, might serve to directly detoxify H 2 O 2 in living organisms. H 2 O 2 and melatonin are present in all subcellular compartments; thus, presumably, one important function of melatonin may be complementary in function to catalase and glutathione peroxidase in keeping intracellular H 2 O 2 concentrations at steady-state levels.