Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.