The proinflammatory cytokines, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and interferon gamma (IFNgamma), are cytotoxic to pancreatic islet beta cells, possibly by inducing nitric oxide and/or oxygen radical production in the beta cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet beta-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1beta, TNFalpha, and IFNgamma. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet beta cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet beta-cell destruction. L-NG-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet beta-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet beta-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet beta cells.