Objective: In vascular biology, endothelial K(Ca)2.3 and K(Ca)3.1 channels contribute to arterial blood pressure regulation by producing membrane hyperpolarization and smooth muscle relaxation. The role of K(Ca)2.3 and K(Ca)3.1 channels in the pulmonary circulation is not fully established. Using mice with genetically encoded deficit of K(Ca)2.3 and K(Ca)3.1 channels, this study investigated the effect of loss of the channels in hypoxia-induced pulmonary hypertension. Approach and Result: Male wild type and K(Ca)3.1(-/-)/K(Ca)2.3(T/T(+DOX)) mice were exposed to chronic hypoxia for four weeks to induce pulmonary hypertension. The degree of pulmonary hypertension was evaluated by right ventricular pressure and assessment of right ventricular hypertrophy. Segments of pulmonary arteries were mounted in a wire myograph for functional studies and morphometric studies were performed on lung sections. Chronic hypoxia induced pulmonary hypertension, right ventricular hypertrophy, increased lung weight, and increased hematocrit levels in either genotype. The K(Ca)3.1(-/-)/K(Ca)2.3(T/T(+DOX)) mice developed structural alterations in the heart with increased right ventricular wall thickness as well as in pulmonary vessels with increased lumen size in partially-and fully-muscularized vessels and decreased wall area, not seen in wild type mice. Exposure to chronic hypoxia up-regulated the gene expression of the K(Ca)2.3 channel by twofold in wild type mice and increased by 2.5-fold the relaxation evoked by the K(Ca)2.3 and K(Ca)3.1 channel activator NS309, whereas the acetylcholine-induced relaxation - sensitive to the combination of K(Ca)2.3 and K(Ca)3.1 channel blockers, apamin and charybdotoxin - was reduced by 2.5-fold in chronic hypoxic mice of either genotype. Conclusion: Despite the deficits of the K(Ca)2.3 and K(Ca)3.1 channels failed to change hypoxia-induced pulmonary hypertension, the up-regulation of K(Ca)2.3-gene expression and increased NS309-induced relaxation in wild-type mice point to a novel mechanism to counteract pulmonary hypertension and to a potential therapeutic utility of K(Ca)2.3/K(Ca)3.1 activators for the treatment of pulmonary hypertension.