Under normoxic conditions in vitro, isolated pulmonary arteries from broilers exhibit reduced endothelium-dependent relaxation responses when compared with Leghorns. In vivo, hypoxia increases the susceptibility of broiler chickens to pulmonary hypertension syndrome (PHS), whereas Leghorns are considered resistant to PHS. Because L-arginine supplementation decreases the incidence of PHS in vivo and improves the relaxation responses of broiler isolated pulmonary arteries in vitro, we hypothesized that in vitro hypoxia would further reduce the relaxation responses of broilers to endothelium-derived nitric oxide (EDNO)-dependent vasodilators and that L-arginine supplementation would alleviate this impairment. As a test of this hypothesis, pulmonary arteries from broiler and Leghorn chickens were isolated and exposed to normoxia or hypoxia in the presence or absence of L-arginine while their constriction and relaxation responses to vasoactive compounds were recorded. In broilers, hypoxia did not affect the constriction responses of isolated pulmonary arteries but decreased EDNO-dependent acetylcholine-induced relaxation responses. In contrast, in Leghorns hypoxia increased endothelin-1-induced vasoconstriction responses and reduced the EDNO-dependent relaxation responses only to the lowest concentration of acetylcholine used. L-Arginine supplementation augmented the relaxation responses to acetylcholine in broilers and Leghorns under normoxia but failed to augment them under hypoxia. Relaxation responses to the NO donor, sodium nitroprusside, were not affected by hypoxia in Leghorns but were increased by hypoxia in broilers. These results suggest that the increased incidence of PHS in broiler chickens reared under hypoxia may be associated with a hypoxia-induced reduction in the synthesis or activity of EDNO in the pulmonary circulation.