Site-specific DNA damage by Cu(II) plus H2O2 was investigated by a DNA-sequencing technique. Cu(II) plus H2O2 induced strong DNA cleavage even without piperidine treatment. Piperidine-labile sites were induced frequently at thymine and guanine residues and rarely at adenine residue. A Cu(I)-specific chelating agent, bathocuproine, inhibited the DNA damage. Neither ethanol nor mannitol inhibited it. Of alcohols, tertbutyl alcohol, having relatively low reactivity to hydroxyl free radical, inhibited the DNA damage most strongly. Sodium azide and 1,4-diazobicyclo[2.2.2]octane completely inhibited cleavages at residues of the bases other than guanine. Tris inhibited the DNA damage. The enhancing effect of D2O on DNA damage was not observed. ESR studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) showed that the hydroxyl radical adduct of DMPO was formed during the reaction of Cu(II) with H2O2, and that the addition of sodium formate produced the CO2- radical adduct of DMPO more efficiently than expected. ESR studies showed that the nitroxide radical was formed from 2,2,6,6-tetramethyl-4-piperidone in the presence of Cu(II) plus H2O2, indicating the formation of singlet oxygen or its equivalent. The effects of scavengers on DNA damage have considerable correlation with the effects of scavengers on the nitroxide radical production and DMPO.OH formation. The results suggest that the main active species causing DNA damage are more likely copper-peroxide complexes, with similar reactivity to singlet oxygen and/or hydroxyl radical rather than hydroxyl free radical.