eNOS expression and activity are decreased in fetal lambs with persistent pulmonary hypertension (PPHN). We sought to determine the impact of mechanical ventilation with O₂ with or without inhaled nitric oxide (iNO) or recombinant human superoxide dismutase (rhSOD) on eNOS in the ductal ligation model of PPHN. PPHN lambs and age-matched controls were ventilated with 100% O₂ for 24 hours alone or combined with either 20 ppm iNO continuously or a single dose of rhSOD (5 mg/kg) given intratracheally at delivery. In one-day, spontaneously breathing lambs (1DSB), eNOS expression in resistance pulmonary arteries (PA) increased relative to fetal levels. eNOS expression increased in control lambs ventilated with 100% O₂, but not in PPHN lambs. Addition of either iNO or rhSOD increased eNOS expression and decreased the generation of reactive oxygen species (ROS) in PPHN lambs relative to those ventilated with 100% O₂ alone. However, only rhSOD restored eNOS function, increased BH4, a critical co-factor for eNOS function, and restored GTP cyclohydrolase I expression in isolated vessels and lungs from PPHN lambs. These data suggest that ventilation of PPHN lambs with 100% O₂ increases ROS production, blunts postnatal increases in eNOS expression, and decreases available BH4 in PPHN lambs. While the addition of either iNO or rhSOD diminished ROS production and increased eNOS expression, only rhSOD improved eNOS function and levels of available BH4. Thus, therapies designed to decrease oxidative stress and restore eNOS coupling, such as rhSOD, may prove useful in the treatment of PPHN in newborn infants.