Because increased flow and shear stress upregulate endothelial (e) nitric oxide synthase (NOS) in adult endothelial cells in vivo and in vitro, we hypothesized that decreased pulmonary blood flow would decrease eNOS content in the late-gestation ovine fetus. To investigate the effects of decreased blood flow and the potential role of altered eNOS content in lung hypoplasia, we studied an animal model of lung hypoplasia after left pulmonary artery (LPA) ligation in nine fetal lambs (114-124 days gestation; term = 147 days). After at least 14 days, animals were killed, and lungs were harvested for histology, immunostaining, Western blot analysis for eNOS protein content, and biochemical assays of NOS activity. LPA ligation markedly reduced left lung size. Histology demonstrated loose connective tissue and airway immaturity in the left lungs. eNOS immunostaining demonstrated equal staining in the left pulmonary vessels compared with the right. Solitary endothelial cells staining for eNOS and factor VIII-related antigen were observed throughout the mesenchyme of left, but not right, lungs. eNOS protein content and activity were similar in left and right lungs. We conclude that, despite the absence of pulmonary blood flow and marked lung hypoplasia, eNOS content and NOS activity were not reduced after LPA ligation in the late fetal lung. We speculate that low pulmonary blood flow does not downregulate fetal pulmonary vascular eNOS expression and that other factors, such as paracrine or autocrine stimuli, may account for the persistence of eNOS in the developing lung circulation.