Nitric oxide (NO), an important cell messenger molecule, is formed endogenously in the lung airway. Three individual genes of NO synthase (NOS), which represent brain NOS (bNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), have been reported in the cultured lung epithelium. Although studies in vivo showed that bNOS and iNOS were expressed and localized in the cytoplasm of bronchial epithelium, the expression and localization of eNOS remains to be determined. Therefore, we employed an eNOS monoclonal antibody whose immunospecificity was tested by both Western blot and preadsorption immunohistochemistry to immunostain rat lungs from fetus to adult. The results showed that eNOS immunoreactivity began to appear in the lung epithelium within 2 hr after birth. Six hours later (8 hr after birth), the NOS immunoreaction was concentrated near the surface of the ciliated epithelial cells. This staining pattern appeared in lungs at Day 1, Week 1, Week 2, and in adult rats. By electron microscopy, eNOS immunoreactivity was confirmed within ciliated epithelium and was shown to be associated with the basal microtubule membrane of the cilia. Nonciliated cells were not stained. Type II epithelial cells also contain eNOS immunoreactivity, which is primarily associated with rough endoplasmic reticulum, and free ribosomes. However, macrophages in the lungs lacked eNOS immunoreactivity. This study demonstrated that eNOS was postnatally expressed in rat bronchial ciliated epithelium. The localization of eNOS at the basal membrane of ciliary microtubules suggests that eNOS may be involved in the function of epithelial cilia, consistent with previous physiological studies.