The aim of the present study was to investigate the role of poly(ADP-ribose) synthetase in a nonseptic shock model, wherein oxyradicals, nitric oxide, and peroxynitrite are known to play a crucial role in the inflammatory process. DNA single-strand breakage and activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS) triggers an energy-consuming, inefficient repair cycle, which contributes to peroxynitrite-induced cellular injury. Here we investigated whether peroxynitrite production and PARS activation are involved in cytotoxicity in macrophages collected from rats subjected to zymosan-induced shock. Macrophages harvested from the peritoneal cavity exhibited a significant production of peroxynitrite, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123. Furthermore, zymosan-induced shock caused a suppression of macrophage mitochondrial respiration, DNA strand breakage, activation of PARS, and reduction of NAD+ cellular levels. In vivo treatment with 3-aminobenzamide (10 mg/kg intraperitoneally, 1 and 6 h after zymosan injection) or nicotinamide (50 mg/kg intraperitoneally, 1 and 6 h after zymosan injection) significantly inhibited the decrease in mitochondrial respiration and the activation of PARS, and partially restored the cellular level of NAD+. In a separate group of experiments, in vivo pretreatment with NG-nitro-L-arginine methyl ester, a nonselective inhibitor of nitric oxide synthesis (10 mg/kg intraperitoneally, 15 min before zymosan administration), reduced peroxynitrite formation and prevented the appearance of DNA damage, the decrease in mitochondrial respiration, and the loss of cellular levels of NAD+. Our study suggests that formation of peroxynitrite and subsequent activation of PARS may alter macrophage function in inflammatory processes and inhibition of nitric oxide, and that PARS may be a novel pharmacological approach to prevent cell injury in inflammation.