Nitric oxide (NO), derived from airway epithelium, is an important mediator of airway function. We have previously demonstrated that the endothelial isoform of nitric oxide synthase (NOS), or eNOS, is expressed in mature airway epithelial cells. The purpose of the present study was to characterize the cellular distribution of eNOS expression in the developing airway and in other pulmonary cell types. In addition, we sought to compare eNOS localization with that of inducible NOS (iNOS) and neuronal NOS (nNOS). Immunohistochemistry was performed on paraformaldehyde-fixed fetal (125-135 d gestation, term =144 d), newborn (2-4 wk.), and maternal sheep lung using antisera specifically directed against eNOS, iNOS, or nNOS protein. Incubations were performed on all age groups simultaneously, and endothelial cells, tracheal macrophages, and cerebellum served as positive controls for eNOS, iNOS, and nNOS, respectively. In fetal lung, eNOS expression was readily evident in the bronchial and bronchiolar epithelium. eNOS was not detected in terminal or respiratory bronchioles or alveolar epithelium. RT-PCR for eNOS in dissected bronchial epithelium confirmed the identity of the isoform. Similar to eNOS, iNOS expression was observed in the airway epithelium of the fetal bronchus and bronchiole, but not in terminal or respiratory bronchioles or alveolar epithelium. As expected, eNOS was detected in endothelium and iNOS was not. In contrast to eNOS and iNOS, nNOS expression was readily evident in respiratory epithelium at all levels of the fetal airway. Furthermore, nNOS was expressed in both airway and vascular smooth muscle. The cellular distribution of all three isoforms was similar in fetal, newborn, and adult lung. However, the intensity of eNOS staining was greater in adult versus fetal and newborn airway epithelium. In contrast, the intensity of epithelial iNOS and nNOS staining was similar in the three age groups. Thus, eNOS, iNOS, and nNOS are all expressed in proximal airway epithelium, and nNOS is the principal isoform in the distal epithelium and also in pulmonary smooth muscle. In addition, the abundance of epithelial eNOS may be regulated during lung maturation. These findings suggest that all three isoforms are important sources of epithelium-derived NO throughout lung development.