Alveolar macrophages represent the first line of defense of the lung against inhaled environmental agents. These cells release a variety of inflammatory mediators including reactive oxygen and nitrogen intermediates that have been implicated in host defense and in tissue injury. In the present studies we characterized production of these mediators by lung phagocytes after exposure of rats to an inhaled pulmonary irritant. Freshly isolated alveolar macrophages from control rats were found to produce nitric oxide as well as hydrogen peroxide and superoxide anion in response to in vitro treatment with inflammatory mediators such as IFN-gamma or LPS and phorbol esters, respectively. Production of nitric oxide by lung phagocytes was enhanced in the presence of superoxide dismutase. Western blot analysis revealed that production of nitric oxide after treatment of the cells with IFN-gamma and LPS was a result of increased expression of inducible nitric oxide synthase. After brief exposure of rats to ozone (O3, 1 to 2 ppm, 3 h), a pulmonary irritant and inflammatory agent that is rapidly converted to molecular oxygen, lung phagocytes produced significantly increased amounts of nitric oxide when compared with control animals. These cells were also sensitized to produce more nitric oxide in response to in vitro treatment with IFN-gamma and LPS. This was due, at least in part, to increased expression of inducible nitric oxide synthase by the cells, which was evident in protein blots and in immunohistochemically stained sections of lung tissue. In further studies we found that O3 inhalation also caused enhanced production of hydrogen peroxide, but an apparent decrease in release of superoxide anion by lung phagocytes. Taken together, these data demonstrate that acute irritant exposure modifies production of reactive oxygen and nitrogen intermediates by lung phagocytes. These alterations may be important in the pathophysiologic response of the lungs to irritants.