Biomolecule oxidation promoted by Cu, Zn-superoxide dismutase (SOD1) has been studied because of its potential role in neurodegenerative diseases. We studied the mechanism of DNA damage promoted by the SOD1–H₂O₂ system. The system promoted the formation of strand breaks in plasmid DNA and the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in calf thymus DNA. We were also able to detect, for the first time, 1,N²-etheno-2′-deoxyguanosine (1,N²-εdGuo) in calf thymus DNA exposed to SOD1–H₂O₂. The addition of a copper chelator caused a decrease in the frequency of 8-oxodGuo and 1,N²-εdGuo, indicating the participation of copper ions lost from SOD1 active sites. The addition of bicarbonate increased the levels of both DNA lesions. We conclude that copper liberated from SOD1 active sites has a central role in the mechanism of DNA damage promoted by SOD1 in the presence of H₂O₂, and that bicarbonate can modulate the reactivity of released copper.