Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ET_B) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (∼0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ET_B receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po₂) compared with normoxic control (140 mmHg Po₂). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H₂O₂. The hypoxic effect on ET_B receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-l-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H₂O₂, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ET_B receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ET_B receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ET_B receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ET_B receptors on smooth muscle.