Chronic lung injury after premature birth is associated with elevated pulmonary vascular resistance and increased muscularization of vessel walls. Nitric oxide (NO), a potent vasodilator produced in the endothelium by the enzyme nitric oxide synthase (eNOS), is an important mediator of pulmonary vasomotor tone. NO is also known to inhibit smooth muscle growth in vitro. During normal fetal development eNOS expression increases during mid and late gestation. We hypothesized that with premature birth requiring prolonged mechanical ventilation, eNOS expression would be decreased, contributing to elevated PVR and to increased smooth muscle growth. To test this hypothesis, we examined the expression of eNOS protein using immunohistochemistry in an animal model of chronic lung disease. Fetal sheep were delivered at 124± 1 d gestation (term = 147 d) and mechanically ventilated for 3-4 wks. Prolonged mechanical ventilation resulted in histologic changes that closely parallel those seen in autopsy specimens from infants with chronic lung disease: mixed overinflation/atelectasis, inflammation, edema, and increased muscularization of pulmonary vessels. We compared eNOS expression in lungs from these preterm lambs (n = 3) to normal term lambs <24 h old (gestation matched, n = 2). Formalin-fixed, paraffin-embedded tissue sections were used for immunoperoxidase staining with a monoclonal antibody specific for eNOS. Studies were also performed without either the primary or secondary antibody to control for non-specific staining. eNOS expression was readily detectable in vascular endothelium of pulmonary arteries and veins from normal term lambs. There was decreased expression of eNOS in pulmonary endothelium from preterm lambs with chronic lung injury. This decreased expression may contribute to the elevated PVR and increased muscularization seen in chronic lung disease of prematurity. (March of Dimes #6FY95 0981)