The effect of hypoxia on endothelium-dependent and endothelium-independent vasodilation was studied in phenylephrine-precontracted, isolated rings of rabbit first-branch pulmonary artery. Concentration-dependent relaxation responses to the endothelium-dependent dilators methacholine, ATP, and the calcium ionophore (A23187) as well as to the endothelium-independent dilators sodium nitroprusside and isoproterenol were obtained before, during, and after exposure to hypoxia (PO2=42 ± l mm Hg) in the presence of indomethacin (2.8 ×K−5M). This moderate degree of hypoxia inhibited (p<0.05) endothelium-dependent but not endotheliumindependent relaxation responses without producing irreversible vascular damage. In parallel experiments, cyclic GMP accumulation in pulmonary vascular rings in response to maximal doses of the above vasodilators was measured in the presence and absence of hypoxia. Cyclic GMP accumulation in response to endothelium-dependent dilators (methacholine, ATP, and A23187) was inhibited (p<0.05) by hypoxia while cyclic GMP accumulation in response to the endothelium-independent dilator sodium nitroprusside was not. When phenylephrine precontracted vessels were exposed to hypoxia in the absence of vasodilators, a small, transient increase in tension occurred, which was greater in endothelium-intact than hi endotheliumdenuded vessels (0.70 ± 0.12 vs. 0.09 ± 0.03 g, respectively;p<0.01). This increase in tension was reduced in the presence of hemoglobin (l×l0−6M; p<0.01), methylene blue (l × l×−7M; p<0.01), and hydroqulnone (l×l0−6M;<0.01) in endothelium-intact but not in endotheliumdenuded rings. Hypoxia also reduced basal cyclic GMP content in endothelium-intact phenylephrine-precontracted rings (1.23 ± 0.22 vs. 0.79 ± 0.19 pmol/mg protein;p<0.05). These data suggest that the transient vasoconstriction induced by hypoxia in these large pulmonary arteries is due partially to the inhibition of basal EDRF production. The observed pharmacological responses imply that the site of hypoxia-induced inhibition of endothelium-dependent dilation is distal to receptor-mediated events in the endothelial cell and proximal to activation of guanylate cyclase In the vascular smooth muscle.