The toxicity paradigm used in the present study involves exposure of U937 cells to a concentration of authentic peroxynitrite, leading to a rapid necrotic response mediated by mitochondrial permeability transition. We found that addition of catalase after treatment with peroxynitrite specifically prevents the loss of mitochondrial membrane potential and the ensuing lethal response. The protective effects of catalase were mimicked by the cocktail glutathione peroxidase/reduced glutathione. A defensive role of intracellular catalase was implied by experiments showing that catalase-depleted cells are hypersensitive to peroxynitrite and that cells with an increased catalase content, selected for their resistance to H 2 O 2 , are cross-resistant to peroxynitrite. Further experiments demonstrated that H 2 O 2 formation takes place after peroxynitrite exposure. Various approaches using inhibitors of the mitochondrial respiratory chain as well as respiration-deficient cells revealed that the oxidant is produced upon dismutation of superoxides generated at the level of complex III. Interestingly, respiration-deficient cells were found to be resistant to peroxynitrite toxicity, and all those treatments increasing formation of H 2 O 2 produced a parallel increase in toxicity. In conclusion, the results presented in this study indicate that peroxynitrite-induced impairment of electron transport from cytochrome b to cytochrome c1 leads to delayed formation of hydrogen peroxide, which plays a pivotal role in the ensuing necrotic response.Peroxynitrite, the reaction product of nitric oxide (NO) and superoxide, is a potent biological oxidant that mediates tissue injury in diverse pathological conditions, including ischemia-reperfusion injury, immunocomplex-mediated pulmonary edema, acute endotoxemia, neurological disorders, and atherosclerosis (Moncada et al., 1991;Heales et al., 1999). At the cellular level, peroxynitrite causes deleterious effects on various biomolecules; indeed, an extensive literature documents its ability to promote lipid peroxidation (Radi et al., 1991), protein nitration and nitrosylation (Patel et al., 1999), DNA damage (Salgo et al., 1995a;Szabó, 1996;Guidarelli et al., 2000) and oxidation of thiols (Salgo et al., 1995a). Although each of these events, or their combination, can be a cause of important dysfunctions and can lead to apoptosis (Lin et al., 1995;Salgo et al., 1995b;Shin et al., 1996;Szabó, 1996;Lin et al., 1997;Foresti et al., 1999;Oh-hashi et al., 1999;Virá g et al., 1999) and/or necrosis (Delaney et al., 1996), it is unclear whether direct molecular damage is the sole mechanism whereby peroxynitrite causes cell death. This is an important point that needs to be clarified, because prevention of direct effects of peroxynitrite can be achieved only via its scavenging or by inhibiting its formation. Because of the very fast decomposition rate of peroxynitrite (half-life Ͻ 1 s) at physiological pH values (Hughes, 1999), it seems obvious that the potential cytoprotective strategies are ...