Endothellial nitric oxide synthase (eNOS) is present early in gestation in the normal developing lung and modulates pulmonary vascular tone in utero and at birth. Pulmon: hypertension often accompanies neonatal disorders associated with lung hypoplasia, but mechanisms causing altered vascular tone and growth are unknown. Hypoperfusion of the lung due to ligation of the left pulmonary artery (LPA) causes unilateral lung hypoplasia in fetal lambs. Since increased flow and shear stress upregulate eNOS in experimental settings, we hypothesized that decreased blood flow would decrease eNOS content in lung hypoplasia. To investigate the potential role of NO in experimental lung hypoplasia, we studied an animal model of lung hypoplasia after LPA ligation in 7 fetal lambs (114-124 days gestation; 147, term). After at least 21 days, animals were sacrificed and lungs were harvested for histology, immunostaining and western blot analysis for eNOS protein content. LPA ligation markedly reduced lung size and structure of the left but not right lung. Histologic evaluation revealed abnormal morphology of the left lung, including decreased parenchymal area, fibrosis, and airway immaturity. eNOS immunostaining demonstrated selective staining for pulmonary vessels and greater intensity of staining in the left (hypoplastic) lung than the right lung. Solitary endothelial cells which stained intensely for eNOS were observed throughout the mesenchyme in the left lung. Densitometric scans of eNOS western blots demonstrated that eNOS protein content was similar in left and right lungs. We conclude that despite the absence of pulmonary artery blood flow and marked hypoplasia, eNOS content was not reduced in this experimental model of lung hypoplasia. We speculate that low pulmonary blood flow does not down-regulate fetal pulmonary vascular eNOS expression despite marked lung hypoplasia.