Background: Decreased cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride (Cl) secretion across mucosal surfaces contributes to the development of airway disease by depleting airway surface liquid, increasing mucus viscosity and adhesion, and consequently hindering mucociliary clearance. We serendipitously discovered during testing of drugs solubilized in low concentrations ethanol (0.25%, 43 mM) that the control vehicle produced robust activation of CFTR-mediated Cl- transport. The objective of the current study is to investigate low concentrations of ethanol for effects on Cl- secretion and ciliary beat frequency (CBF). Methods: Wild-type (WT) and transgenic CFTR-/- primary murine nasoseptal epithelial (MNSE) cultures and WT and F508del/F508del human sinonasal epithelial (HSNE) cultures were subjected to transepithelial ion transport measurements using pharmacologic manipulation in Ussing chambers. CBF activation was also monitored. Murine nasal potential difference (NPD) was measured in vivo. Results: Ussing chamber tracings revealed ethanol activated CFTR-mediated Cl transport in a dose-dependent fashion in WT MNSE (n = 4, p < 0.05) and HSNE (n = 4, p < 0.05). Ethanol also significantly increased CBF (fold change) in WT MNSE cultures in a dose-dependent fashion (phosphate-buffered saline [PBS], 1.33 ± 0.04; 0.25% ethanol, 1.37 ± 0.09; 0.5% ethanol, 1.53 ± 0.06 [p < 0.05]; 1% ethanol, 1.62 ± 0.1 [p < 0.05]). Lack of stimulation in CFTR-/- and F508del/F508del cultures indicated activity was dependent on the presence of intact functional CFTR. Ethanol perfusion (0.5%) resulted in a significant -3.5-mV mean NPD polarization when compared to control solution (p < 0.05). Conclusion: The observation that brief exposure of ethanol stimulated Cl- secretion via CFTR-mediated pathways indicates its possible use as topical aerosol delivered alone or in combination with other CFTR activators for diseases of dysfunctional mucociliary clearance (MCC) in chronic rhinosinusitis (CRS).