Although oxidative stress is known to contribute to endothelial dysfunction-associated systemic vascular disorders, its role in pulmonary vascular disorders is less clear. Our previous studies, using isolated pulmonary arteries taken from lambs with surgically created heart defect and increased pulmonary blood flow (Shunt), have suggested a role for reactive oxygen species (ROS) in the endothelial dysfunction of pulmonary hypertension, but in vivo data are lacking. Thus the initial objective of this study was to determine whether Shunt lambs had elevated levels of ROS generation and whether this was associated with alterations in antioxidant capacity. Our results indicate that superoxide, but not hydrogen peroxide, levels were significantly elevated in Shunt lambs. In addition, we found that the increase in superoxide generation was not associated with alterations in antioxidant enzyme expression or activity. These data suggested that there is an increase in superoxide generation rather than a decrease in scavenging capacity in the lung. Thus we next examined the expression of various subunits of the NADPH oxidase complex as a potential source of the superoxide production. Results indicated that the expression of Rac1 and p47(phox) is increased in Shunt lambs. We also found that the NADPH oxidase inhibitor diphenyliodonium (DPI) significantly reduced dihydroethidium (DHE) oxidation in lung sections prepared from Shunt but not Control lambs. As DPI can also inhibit endothelial nitric oxide synthase (eNOS) superoxide generation, we repeated this experiment using a more specific NADPH oxidase inhibitor (apocynin) and an inhibitor of NOS (3-ethylisothiourea). Our results indicated that both inhibitors significantly reduced DHE oxidation in lung sections prepared from Shunt but not Control lambs. To further investigate the mechanism by which eNOS becomes uncoupled in Shunt lambs, we evaluated the levels of dihydrobiopterin (BH(2)) and tetrahydrobiopterin (BH(4)) in lung tissues of Shunt and Control lambs. Our data indicated that although BH(4) levels were unchanged, BH(2) levels were significantly increased. Finally, we demonstrated that the addition of BH(2) produced an increase in superoxide generation from purified, recombinant eNOS. In conclusion our data demonstrate that the development of pulmonary hypertension in Shunt lambs is associated with increases in oxidative stress that are not explained by decreases in antioxidant expression or activity. Rather, the observed increase in oxidative stress is due, at least in part, to increased expression and activity of the NADPH oxidase complex and uncoupled eNOS due to elevated levels of BH(2).