Selenite catalytically oxidizes reduced glutathione (GSH) with subsequent generation of superoxide. Our laboratory has previously shown that this selenite-catalyzed generation of superoxide is strongly inhibited by copper [as copper(II) sulfate]. In the present study we have demonstrated that exposure of human colonic carcinoma cells (HT-29) to selenite resulted in the induction of apoptosis, DNA fragmentation, an increase in intracellular levels of the antioxidant GSH, and cytotoxicity. Selenite- induced apoptosis, DNA fragmentation, increases in GSH levels, and cytotoxicity were inhibited by copper(II) sulfate. Copper only protected cells from selenite cytotoxicity when cells were exposed to selenite and copper simultaneously, not when cells were pretreated with copper, then washed before selenite exposure. This suggests that copper elicits its protective effect extracellularly. Previous data reported by this laboratory clearly demonstrated that copper inhibited selenite-catalyzed superoxide generation. Collectively, these data suggest that reactive oxygen species may play a role in selenite-induced cytotoxicity, apoptosis, and DNA fragmentation.