Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. We tested the hypothesis that long-term high altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in eNOS. PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea-level (300 m) or high altitude of 3,801 m for 110 days. Hypoxia had no effect on wall thickness and KCl-induced contractions of pulmonary vessels. In PA, hypoxia increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor L-NNA. In PV, hypoxia had no effect on NE-induced contractions in the absence of L-NNA. L-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of L-NNA, NE-induced contractions of PV were significantly decreased in hypoxic as compared with normoxic lambs. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD2 and the maximal response of acetylcholine-induced relaxation. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high altitude hypoxia and suggest a likely common mechanism downstream of nitric oxide in fetal pulmonary vessel response to chronic hypoxia in utero.