The epithelial sodium channel (ENaC) mediates transepithelial sodium absorption across airway epithelia and contributes to volume homeostasis of airway surface liquid (ASL). Increased ENaC activity leads to ASL depletion and impairment of mucociliary clearance. Bile acids (BA) directly activate ENaC in heterologous expression systems. In cystic fibrosis patients, BA appear in ASL due to gastro-oesophageal reflux and aspiration. We hypothesise that BA contributes to enhanced ENaC activity and ASL depletion in cystic fibrosis airways. We measured the effects of BA on heterologously expressed human ENaCs and endogenous ENaCs in human H441 airway epithelial cells. Human αβγ- and δβγ-ENaCs were expressed in Xenopus oocytes and channel activity was measured by the two-electrode voltage-clamp technique at -60 mV. ENaC-mediated current fractions were determined using the inhibitor amiloride (100 μM). Sodium salts of the BA cholic acid (CA), chenodeoxycholic acid (CDCA), taurocholic acid (t-CA) and taurochenodeoxycholic acid (t-CDCA) were applied to a final concentration of 100 μM. Amiloride-sensitive currents (ΔIami) before and after application of BA were analysed. All data (n = 6-7) were normally distributed and analysed with Student’s paired t-test. Human H441 airway epithelial cells were cultured to polarised monolayers at air/liquid interface and sodium absorption was measured as transepithelial short-circuit current (ISC) in Ussing chambers. ISC signals were baseline corrected (100 µM amiloride) and normalised to values before application of BA. Data (n = 4-5) are reported as means ± standard error. CA significantly increased ΔIami in αβγ-ENaC expressing cells from 3.32 ± 0.55 to 3.61 ± 0.59 µA (p<0.05) and from 8.63 ± 1.42 to 11.33 ± 1.68 µA in oocytes expressing δβγ-ENaC (p<0.05). CDCA had no effect on αβγ-ENaC (p = 0.15) but increased ΔIami from 8.45 ± 2.03 to 11.21 ± 2.60 µA in oocytes expressing δβγ-ENaC (p<0.05). t-CA significantly increased ΔIami from 3.68 ± 0.66 to 4.72 ± 0.77 µA (p<0.001) in αβγ-ENaC and from 6.8 ± 0.95 to 8.54 ± 1.12 µA in δβγ-ENaC expressing cells (p<0.001). t-CDCA significantly increased ΔIami in αβγ-ENaC expressing cells from 4.87 ± 0.48 to 6.37 ± 0.71 µA (p<0.05) and from 6.21 ± 0.57 to 8.64 ± 0.76 µA in oocytes expressing δβγ-ENaC (p<0.001). In H441 monolayers, t-CDCA significantly increased ISC by 9 ± 0.02 % after 2 min of application (p = 0.036; RM one-way ANOVA/Dunett’s multiple comparisons test), whereas no other BA had any effect on ISC. After 5 min of exposure to any BA, there were no differences in normalised ΔIami (p=0.25; one-way ANOVA). These data indicate that BA have the capacity to activate human ENaC in heterologous expression systems. There is no such effect on endogenous ENaC in human H441 airway epithelial cells. This discrepancy might be due to differences in ENaC maturation in heterologous expression systems compared to cultured airway epithelial cells.