Male cystic fibrosis (CF) patients survive 9 years longer than females (“CF gender gap”) and lung exacerbations in CF females vary during the estrous cycle. Estrogen has been reported to reduce the height of the airway surface liquid (ASL) in female CF bronchial epithelium. Here we investigated the effect of estrogen on ASL height and ion transport in normal (NuLi-1) and CF (CuFi-1) bronchial epithelium monolayers grown on permeable filters in an air-liquid interface. Confocal fluorescence microscopy experiments were performed to measure ASL height in normal and CF bronchial epithelial cells treated with 17β-estradiol (E2 0.1 to 10nM). In order to determine the ion channel(s) involved in the effect of E2 on ASL height, different ion transporter modulators were used in ASL and Ussing chamber experiments. The nuclear-excluded Estrogen Dendrimer Conjugate and its negative control (empty dendrimer) were used in order to determine the involvement of the nuclear estrogen receptor pathway in the effect of E2 on ASL height. Confocal experiments revealed that ASL height was significantly higher in the non-CF cell line compared to the CF cells (NuLi 6.82 ± 0.33μm vs. CuFi 5.58 ± 0.14μm, n=20, p<0.001). Physiological concentrations of E2 reduced the ASL height in both non-CF (25% decrease, n=5, p<0.05, ANOVA) and CF (20% decrease, n=5, p<0.05, ANOVA) cell lines after 30 min treatment. Treatment with the Cl- transport inhibitor bumetanide or the KCNQ1 potassium channel blocker chromanol HMR1556 decreased ASL height significantly in both cell lines. However, E2 had no additive effect on ASL height in the presence of these ion transporter inhibitors. E2 decreased the bumetanide-sensitive Cl- current in normal cells (E2: 6.47 ± 2.08 μA/cm2, Control: 9.52 ± 2.08 μA/cm2, n=3, p<0.05, paired t-test) and produced an increase in amiloride sensitive current in CF cells (E2: 11.097 ± 1.805 μA/cm2, Control: 8.801 ± 1.464 μA/cm2, n=7, p<0.05, paired t-test). Treatment with the nuclear-impeded Estrogen Dendrimer Conjugate (EDC 0.1 – 1nM E2 equivalent concentration) produced a significant reduction in ASL height in CF and non-CF cells (4.72 ± 0.25μm in NuLi-1 and 4.86 ± 0.42μm in CuFi-1, n=5, p< 0.05 compared to control, ANOVA) whereas the empty dendrimer had no effect. These results demonstrate that E2 dehydrates both CF and normal ASL and these rapid responses to E2 are membrane-initiated rather than via the classical nuclear estrogen receptor signal transduction pathway. The ion transporter inhibitor data indicate that E2 acts on ASL by inhibiting Cl- secretion in non-CF cells and increasing Na+ absorption via ENaC in CF cells.