The inducible nitric oxide (NO) synthase gene in alveolar macrophages (AMs) is a stress response gene that may contribute to tissue injury in the lung after respiration with high O(2) concentrations through extensive production of NO. In this study, we investigated the influence of hyperoxia on the NO pathway in rat AMs in vitro, its regulation by the transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1, and the role of reactive oxygen species (ROS). AMs were treated with lipopolysaccharide (LPS) and/or interferon (IFN)-gamma and incubated under 21 or 85% O(2). Stimulation with LPS and IFN-gamma led to induction of the NO pathway that was further upregulated by hyperoxia. The binding activity of NF-kappaB, in contrast to that of AP-1, was activated on stimulation with LPS and IFN-gamma, and both were further increased under hyperoxia. The antioxidants pyrrolidine dithiocarbamate and N-acetyl-L-cysteine inhibited intracellular ROS production and the NO pathway under both normoxic and hyperoxic conditions but had diverse effects on the transcription factors. The results presented here indicate that hyperoxia can upregulate the NO pathway in stimulated AMs through increased production of intracellular ROS and activation of NF-kappaB and AP-1.