Alterations in nitric oxide signaling have been hypothesized to have an etiologic role in the development of hypoxic pulmonary hypertension. However, changes in the expression of nitric oxide synthase (NOS) in hypoxic lungs remains controversial. In this study, we used (1) Northern and Western analyses to measure NOS mRNA and protein expressions, (2) lung histology together with measurements of lung and heart weights to monitor pulmonary vascular remodeling, and (3) immunohistochemistry to localize NOS proteins. The data demonstrated that endothelial NOS mRNA and protein were upregulated over 1 to 7 days of hypoxia that temporally correlated with and preceded the vascular remodeling that occurred in the course of the development of hypoxic pulmonary hypertension. Hypoxia also induced brain NOS in bronchial epithelium and inducible NOS in vascular smooth muscle but did not affect inducible NOS expression in macrophages or basal guanylyl cyclase activity in the lung. These findings showed that upregulation of endothelial NOS was tightly correlated with the vascular remodeling induced by hypoxia, suggesting a role for nitric oxide in the development of pulmonary hypertension. (Hypertension. 1996;28:743-753.)