H2O2 is known to be capable of inducing strand-break damage in intracellular DNA, but whether O2- also can do so in the absence of H2O2 is uncertain. The difficulty in distinguishing the effects of the two is that, under physiological conditions, dismutation of O2- to H2O2 can readily occur. When human leukocytes are stimulated with phorbol 12-myristate 13-acetate (PMA), they release O2-, and within a few minutes strand breakage in intracellular DNA can be observed. We have attempted to determine whether the O2- produced is itself capable of causing DNA damage or whether H2O2 alone, or in combination with O2-, is responsible for the observed damage. Addition of catalase (up to 250 micrograms/ml) to remove H2O2 prevented no more than about 50% of the DNA damage. The majority of the remaining damage could be blocked, in a dose-dependent manner, by superoxide dismutase (SOD) or a SOD-mimetic copper complex, identifying a fraction of damage to intracellular DNA dependent upon extracellular O2-. We studied this O2(-)-specific fraction through the use of three metabolic poisons (fluoride, 2-deoxyglucose, and A23187). These agents largely blocked DNA damage, while affecting extracellular O2- levels only slightly. For comparison, H2O2-induced DNA damage was studied with glucose oxidase to generate a flux of H2O2. The first two metabolic poisons had little effect, whereas A23187 did inhibit H2O2-induced DNA damage. We conclude that O2(-)-induced damage occurs through a mechanism that differs, at least in part, from the H2O2 damage pathway and that the former may involve one or more metabolic steps.