Nitric oxide synthase (NOS) produces nitric oxide, a mediator of potential importance in numerous physiologic and inflammatory processes in the lung. We localized constitutive NOS (c-NOS) and inducible NOS (i-NOS) within lung tissue by immunoperoxidase labeling with specific antibodies or by histochemical demonstration of the characteristic NADPH diaphorase activity of NOS. We analyzed human airway (n = 4) or parenchyma (n = 10) specimens obtained from uninvolved areas of surgical tumor resections. We also studied human fetal lung samples (n = 6) and normal or inflamed (16 h after intratracheal LPS instillation) rat lung tissue. Immunostaining with anti-c-NOS identified c-NOS antigen in rat lung nerves, endothelium, and airway epithelium. Normal or inflamed rat macrophages were not stained. Human nerve elements and large-vessel endothelium showed immunostaining with the anti-c-NOS, but no labeling of the airway or alveolar epithelium was seen. Immunostaining with anti-i-NOS showed strong labeling of rat macrophages after LPS treatment, in vivo or in vitro, while normals were negative. Human alveolar macrophages were occasionally positive for i-NOS, especially in areas of chronic inflammation, which also showed focal immunolabeling of endothelium. Uniform labeling of epithelium in large, cartilaginous airways was found with anti-i-NOS in both human bronchi and normal rat trachea samples, suggesting a constitutive role for a NOS that shares epitope(s) with or is highly homologous to the inducible, macrophage type of NOS. Histochemical staining for NADPH diaphorase activity was consistent with immunolocalization of NOS antigens.(ABSTRACT TRUNCATED AT 250 WORDS)