An enzymic flux of O2- and H2O2 caused strand breaks in the supercoiled covalently closed circular Col El plasmid. Subnanomolar levels of superoxide levels of superoxide dismutase or of catalase prevented this attack on DNA, signifying that both O2- and H2O2 were required. Benzoate, mannitol or histidine, which do not scavenge O2- or H2O2, also protected the DNA suggesting that the proximate attacking species had a reactivity comparable to that of the hydroxyl radical. Replacing EDTA with diethylene triamine pentaacetic acid eliminated this attack. In toto the data suggest a metal-catalyzed interaction between O2- and H2O2 which generates a potent oxidant, possibly OH, which can cause DNA strand scission. The biological implications of the production and the enzymic scavenging of the superoxide radical are discussed.