Apoptosis and necrosis are two distinct forms of cell death that can occur in response to various agents. In the present study the HepG2 cell line was used for a comparative study of CD95-mediated apoptosis and menadione-induced necrosis. Apoptosis coincided with the release of cytochrome c from mitochondria, activation of caspases, cleavage of cellular proteins, and also involved nuclear condensation and DNA fragmentation. Necrosis was not accompanied by DNA fragmentation, caspase activation or cleavage of caspase target proteins, despite cytochrome c release from mitochondria. In fact, the addition of menadione to cells undergoing CD95-mediated apoptosis blocked their caspase activity. Inhibition of caspases coincided with an accumulation of reactive oxygen species (ROS) and ATP depletion. In order to determine the predominance of either of these events in the inhibition of caspase, cells were either co-incubated with antioxidant enzymes or their ATP level was manipulated to maintain it at a relatively high level during the experiments. Co-incubation with catalase, but not Cu/Zn superoxide dismutase, substantially reduced the levels of ROS and reversed the inhibitory effect of menadione on caspase activity. In contrast, increasing cellular ATP level had little effect on restoring caspase activity. These data suggest that menadione inhibits caspase activity by the generation of hydrogen peroxide through redox cycling and that caspase inactivation by this mechanism may prevent cell death by apoptosis in this oxidative-stress model.