Chronic hypoxia (CH) is a common cause of pulmonary hypertension (PH). Accumulating evidence suggests that changes in the activity of endothelin (ET)-1 receptors may play an important role in CH-induced PH. After 3 weeks of CH (10% O2) exposure, we found that the isolated intra-pulmonary artery (PA) constrictor response to ET-1 was significantly increased in wild-type (wt) mice. The administration of Cu/Zn superoxide dismutase (SOD) markedly reduced the CH-enhanced maximal PA constrictor response to ET-1, demonstrating the contribution of superoxide to CH-enhanced PA constrictor responses. Using mice that are completely deficient in gp91phox (a subunit protein of the superoxide producing nicotinamide adenine dinucleotide phosphate [NADPH] oxidase), we found that CH-enhanced PA constriction to ET-1 was completely blocked (decreases in mean [± SE] maximal isometric tension from 5.43 ± 0.35 to 3.33 ± 0.19 mN; n = 7; p < 0.01). Using a lucigenin-enhanced chemiluminescence technique to measure superoxide, we found that the 3 weeks of CH significantly increased superoxide levels in PA isolated from wt mice. The addition of ET-1 further increased superoxide production. To demonstrate that the increased chemiluminescence is due to superoxide generation, we added Cu/Zn SOD, which markedly decreased chemiluminescence, demonstrating the specificity of this assay. When gp91phox knockout mice were exposed to CH, they had significantly reduced levels of superoxide compared to CH-treated wt mice. Our results demonstrate that the CH-enhanced PA constrictor response to ET-1 is mediated by NADPH oxidase (gp91phox)-derived superoxide overproduction that may contribute to the pathogenesis of CH-induced PH.