We hypothesized that endothelial BKCa channel activation can improve pulmonary endothelial function by causing change in membrane potential (Em), [Ca2+]i and NO release.MethodsLung microvascular endothelial cell (EC) Em, [Ca2+]i and NO change was measured using fluorescence imaging with DiBAC4(3), Fura‐2AM and DAF‐FM, respectively. Phenylephrine preconstricted pulmonary arterial (PA) rings were used to assay the effect of acetylcholine (Ach) on vessel relaxation. PA pressures were monitored in preconstriced isolated ventilated‐perfused rat lungs (IPL) after BKCa channels activation.ResultsBKCa channels were demonstrated in macro‐ and microvascular endothelium by immunohistochemistry in rat lungs and by immunoblot in EC lysates. BKCa channel activation with 30μM NS1619 led to endothelial hyperpolarization and an increase in [Ca2+]i and NO. Also, NS1619 caused enhanced endothelium‐dependent dilation in PA rings, with no effect on endothelium‐independent dilation using NO donor or endothelium‐denuded rings. K+ channel blocker, TEA, abolished the effect of NS1619 on endothelium dependent dilation. In IPL, BKCa channel activation by NS1619 caused a dose‐dependent reduction in PA pressures that was attenuated by the inhibition of NO production.ConclusionPulmonary EC express BKCa channels. Activation of pulmonary endothelial BKCa channels leads to improvement in endothelial function.