We have examined the effects of exposing rats to hypoxia (10% fractional inspired O2 concentration) for 2 and 7 days on endothelium-dependent and -independent vasodilation and also on the ability of guanosine 3',5'-cyclic monophosphate (cGMP) to activate cGMP-dependent protein kinase (G-kinase) in rat conduit pulmonary arteries (PA). The ability of acetylcholine (ACh) and sodium nitroprusside (SNP) to both relax PA rings and elevate tissue cGMP levels was significantly attenuated in PA from hypoxic animals. The ability of atrial natriuretic peptide to relax and generate cGMP in PA rings was unchanged by hypoxia. Relaxation and elevation of cGMP levels induced by SNP in aortic rings was unaltered by hypoxia. Similarly, hypoxia did not alter the concentration-dependent activation by exogenous cGMP of G-kinase. We conclude that chronic exposure of rats to hypoxia results in a selective impairment of soluble guanylyl cyclase in rat PA, leading to an attenuation of ACh- and SNP-induced cGMP accumulation and relaxation.