Inhalation of the pulmonary irritant ozone is associated with an accumulation of macrophages in the lung. These cells, along with type II epithelial cells, are activated to release increased quantities of hydrogen peroxide and nitric oxide, two reactive mediators that have been implicated in tissue injury. In the present studies we determined whether pretreatment of rats with bacterially derived endotoxin, which modulates oxidant levels in tissues, could abrogate the effects of ozone on lung injury and nitric oxide production. Acute exposure of rats to ozone (2 parts per million, 3 h) resulted in nitric oxide production in the lung as measured by electron paramagnetic resonance spin trapping. This was correlated with expression of inducible nitric oxide synthase (iNOS) mRNA in the lung as determined by in situ hybridization. Particularly high levels of iNOS were evident in alveolar macrophages and type II cells. Alveolar macrophages isolated from ozone-treated rats also expressed increased iNOS mRNA and protein as measured by Northern and Western blotting, respectively, and produced more nitric oxide compared with cells from air-exposed animals. Treatment of rats with endotoxin (5 mg/kg, intravenously), 30 min prior to ozone, was found to abrogate ozone-induced increases in iNOS mRNA and protein expression, as well as nitric oxide production by alveolar macrophages. This was associated with a reduction in ozone-induced tissue injury as determined by levels of lung lavage fluid protein. Ozone inhalation also resulted in a reduction in intracellular glutathione in alveolar macrophages, an effect that was blocked by endotoxin administration. Taken together, these data provide evidence that the protective effects of endotoxin against ozone-induced injury are mediated, at least in part, by alterations in levels of lung oxidants and antioxidants.