We investigated whether in vivo melatonin treatment inhibits cellular injury induced by peroxynitrite production and PARS activation 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 suppressed macrophage mitochondrial respiration, DNA strand breakage, activation of the nuclear enzyme poly(ADP-ribose)synthetase (PARS) and reduction of cellular levels of NAD+. In vivo treatment with melatonin (25 and 50 mg/kg, i.p., 1 h after zymosan injection) significantly and dose-dependently reduced peroxynitrite formation and prevented the appearance of DNA damage, decrease in mitochondrial respiration, loss of cellular levels of NAD+ and PARS activation. Our study supports the view that the antioxidant and anti-inflammatoy effect of melatonin is also correlated with the inhibition of peroxynitrite production and PARS activation. In conclusion, melatonin may be a novel pharmacological approach to prevent cell injury in inflammation.