Our main objective was to determine whether reactive oxygen species (ROS), such as superoxide (O₂^.-) and hydrogen peroxide (H₂O₂), contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the cell permeable superoxide dismutase mimetic (SOD, M40403) and/or PEG-Catalase (PEG-CAT) on responses to acetylcholine (ACh) were measured in endothelium-intact and denuded pulmonary resistance arteries (PRAs, 90-300 µm diameter). To determine whether NADPH oxidase is an enzymatic source of ROS, PRA responses to ACh were measured in the presence and absence of an NADPH oxidase inhibitor, apocynin (APO). A western blot technique was used to assess expression of the NADPH oxidase subunit, p67 phox. A lucigenin-derived chemiluminescence technique was used to measure ROS production stimulated by the NADPH oxidase substrate, NADPH. ACh responses, which were dilation in intact control arteries but constriction in both intact and denuded hypoxic arteries, were diminished by M40403, PEG-CAT, the combination of M40403 plus PEG-CAT, as well as by APO. Although total amounts were not different, membrane associated p67phox was greater in PRAs from hypoxic compared to control piglets. NADPH-stimulated lucigenin luminescence was nearly doubled in PRAs from hypoxic versus control piglets. We conclude that ROS generated by NADPH oxidase contribute to the aberrant pulmonary arterial responses in piglets exposed to 3 days of hypoxia.