Calcium-activated potassium channels of small (KCa2, SK) and intermediate (KCa3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of KCa2 and KCa3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The KCa channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by L-nitro-arginine, L-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n= 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na+,K+-ATPase with ouabain or blocking KCa2 and KCa3.1 channels reduced the persisting ACh-induced relaxation. In the presence of L-NOARG and indomethacin, a novel KCa2 and KCa3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of KCa2.3 and KCa3.1 were unaltered, whereas KCa2.3 protein expression was upregulated and KCa3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of KCa2.3 channels. Since impaired EDH-type relaxation was accompanied by KCa3.1 channel protein downregulation, these findings suggest that KCa3.1 channels are important for the maintenance of EDH-type relaxation.