Melatonin, an indolic pineal hormone, is produced primarily at night in mammals and is important in controlling biological rhythms. Although melatonin is known to be effective as a free radical scavenger and has an anti-cancer effect, carcinogenic properties have also been reported. In relation to its carcinogenic potential, we have examined whether 6-hydroxymelatonin, a major melatonin metabolite, can induce DNA damage in the presence of metal ion using [32P]-5'-end-labeled DNA fragments obtained from genes relevant to human cancer. 6-Hydroxymelatonin induced site-specific DNA damage in the presence of Cu(II). Formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at G residues of the 5'-TG-3' sequence, whereas piperidine treatment induced cleavage sites at T mainly of 5'-TG-3'. Interestingly, 6-hydroxymelatonin strongly damaged G and C of the 5'-ACG-3' sequence complementary to codon 273 of the p53 gene. These results suggest that 6-hydroxymelatonin can cause double-base lesions. DNA damage was inhibited by both catalase and bathocuproine, Cu(I)-specific stabilizer, suggesting that reactive species derived from the reaction of H2O2 with Cu(I) participate in DNA damage. Cytochrome P450 reductase efficiently enhanced 6-hydroxymelatonin-induced oxidative DNA damage and oxygen consumption, suggesting the formation of redox cycle. It is noteworthy that 6-hydroxymelatonin can efficiently induce DNA damage via non-o-quinone type of redox cycle. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in calf thymus DNA was significantly increased by 6-hydroxymelatonin in the presence of Cu(II). Furthermore, 6-hydroxymelatonin significantly increased the formation of 8-oxodG in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H2O2)-resistant cell line derived from HL-60. The 6-hydroxymelatonin-induced 8-oxodG formation in HL-60 cells significantly decreased by the addition of bathocuproine or o-phenanthroline. Therefore, it is concluded that melatonin may exhibit carcinogenic potential through oxidative DNA damage by its metabolite.