Expression and localization of nitric oxide synthase (NOS) in the lungs of chronically hypoxic and normoxic rats were studied using both immunohistochemistry and NADPH diaphorase (NADPH-d) staining techniques. In the normoxic and in the hypoxic rat, NOS was detected by both methods in the endothelium of large pulmonary vessels and in the epithelium of bronchi and bronchioli. NOS expression was not detected in the endothelium of normoxic pulmonary resistance vessels but was prominently expressed in the endothelium of these vessels after 2-4 wk of chronic hypoxia. In contrast to small pulmonary vessels, the endothelium of small bronchial vessels exhibited NOS immunostaining in both normoxic and hypoxic lungs. Hypoxia was also found to induce de novo NOS expression in the smooth muscle of large and small pulmonary vessels and in bronchial smooth muscle. NOS enzyme activity in lung homogenates was assessed by [3H]arginine to [3H]citrulline conversion. The activity of soluble NOS, but not particulate NOS, was increased in the hypoxic lungs. These results demonstrate chronic hypoxia-induced upregulation of NOS protein expression and activity in the rat lung, suggesting a potentially important role of nitric oxide in adaptation of the pulmonary circulation to chronic hypoxia. The lack of immunostaining in small pulmonary resistance vessels is also consistent with physiological studies suggesting that NO may not be involved in the mechanism for maintaining the normally low pulmonary vascular resistance.