Reactive oxygen species have been implicated in the gastrointestinal pathogenesis of septic and endotoxic shock. The objective of this study was to investigate the role of inducible nitric oxide synthase during endotoxin-induced formation of oxidants by cells of the small intestine. After intravenous Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) injection, nitric oxide production was measured as nitrosyl complex formation in the ileum using electron paramagnetic resonance spectroscopy. Oxidative stress biomarkers were determined as duodenal mucosal-reduced thiols, the ileal lipid peroxidation and luminal free radical production using spin trapping methodology. Demonstration of nitrosyl complex formation commenced at 3 h and diminished 24 h post-LPS. Mucosal thiol levels were decreased at 3, 6, 12, and 18 h post-LPS treatment. At these time point, the ileal lipid peroxidation also increased as did luminal formation of hydroxyl radical adduct. Nitric oxide synthase inhibitors reversed the elevation of hydroxyl radical formation and reversed the decrease in mucosal-reduced thiol levels in the LPS-treated rats. Our data indicate that nitric oxide or its oxidant product(s), such as peroxynitrite, contribute to oxidative injury in the small intestine of rats treated with endotoxin.