Active Na+ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and KATP K+ channel activities exerts sustained control in Na+ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the alpha-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K+ channels, and 2) to determine the physiological impact of K+ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and KATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24 h) increased alpha-ENaC expression, similarly to KATP activation by pinacidil. Conversely, pharmacological KvLQT1 and KATP inhibition or silencing with siRNAs down-regulated alpha-ENaC expression. Furthermore, K+ channel blockers significantly decreased alpha-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and KATP activation dose-dependently enhanced alpha-ENaC promoter activity. Finally, we noted a physiological impact of changes in K+ channel functions on ERK activity, alpha-, beta-, gamma-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K+ channels regulate alpha-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.