In a recent study (AJP 286:L1027-L1037, 2004), we identified a K_ATP channel in alveolar epithelial cells, formed by Kir6.1/SUR2B subunits. We found that short applications of K_ATP, KvLQT1 and K_Ca channel modulators modified Na⁺ and Cl^- currents in alveolar monolayers. In addition, it was shown previously that a K_ATP opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K⁺ channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24-h with inhibitors of K_ATP, KvLQT1 and K_Ca channels, identified by PCR. Glibenclamide and clofilium (K_ATP and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na⁺ current and forskolin-activated Cl^- currents, whereas pinacidil, a K_ATP activator, increased them. Interestingly, K⁺ inhibitors or membrane depolarization (induced by valinomycin in high-K⁺ medium) decreased -, -, -ENaC and CFTR mRNA. -ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na⁺ transport through ENaC, we tested the impact of K⁺ channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K⁺ channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.