Respiratory distress syndrome (RDS) secondary to preterm birth and surfactant deficiency is characterized by severe hypoxemia, lung injury, and impaired production of nitric oxide (NO) and vascular endothelial growth factor (VEGF). Since hypoxia-inducible factors (HIFs) mediate the effects of both NO and VEGF in part through regulation by prolyl-hydroxylase containing domains (PHDs) in the presence of oxygen, we hypothesized that HIF-1 and -2 in the lung are decreased following severe RDS in preterm neonatal lambs. To test this hypothesis, fetal lambs were delivered at preterm gestation (115 days gestation, term=145 days; n=4), and mechanically ventilated for four hours. Lambs developed respiratory failure characterized by severe hypoxemia despite treatment with mechanical ventilation with high inspired oxygen concentrations. Lung samples were compared with non-ventilated control animals at preterm (115 days gestation; n=3) and term gestation (142 days gestation; n=3). We found that HIF-1 protein expression decreased (p<0.05) and PHD-2 expression increased (p<0.005) at birth in normal term animals prior to air breathing. In comparison with age-matched controls, HIF-1 protein and HIF-2 protein expression decreased by 80% and 55%, respectively (p<0.005 for each) in preterm lambs with RDS. Furthermore, VEGF mRNA was decreased by 50% and PHD-2 protein expression doubled in RDS lambs. We conclude that pulmonary expression of HIF-1, HIF-2, and the downstream target of their regulation, VEGF mRNA, is impaired following RDS in neonatal lambs. We speculate that early disruption of HIF and VEGF expression after preterm birth and RDS may contribute to long-term abnormalities in lung growth, leading to BPD.