SLC26A9, a member of the SLC26 family of anion transporters, has been recently identified to function as an epithelial chloride channel. Experiments using Slc26a9-deficient mice revealed that the Cl- channel was activated in airway inflammation and Cl- secretion was essential for preventing airway obstruction in inflammatory airway disease (1). Hence, activation of SLC26A9-mediated secretion of Cl- may be a promising therapeutic strategy for airway diseases such as cystic fibrosis. However, the underlying molecular mechanism of activation of SLC26A9 at the epithelium remains unknown.Fisher rat thyroid (FRT) cells were used as an in vitro epithelial cell model and transduced with a HA-tagged SLC26A9 construct using retroviral transfection. Expression of SLC26A9 was analysed by RT-PCR and immunocytochemistry. Transepithelial Cl- currents were measured by applying a Cl- gradient under isotonic (290 mosmol/l), hypotonic (220 mosmol/l) and hypertonic conditions (380 mosmol/l) in Ussing chambers. SLC26A9 was highly expressed in transduced FRT (FRT-SLC) cells, which was confirmed by RT-PCR as well as immunocytochemistry. Interestingly, SLC26A9 was predominantly detected intracellularly. Bioelectric measurements revealed that basal short circuit current (Isc) was significantly increased in FRT-SLC (12.3±2.0 µA/cm2) compared to control-transfected FRT (FRT-CTL; 3.9±0.5 µA/cm2) cell monolayers (P<0.05). Further, IBMX/Forskolin-stimulated Cl- secretion was significantly increased in FRT-SLC compared to FRT-CTL cell monolayers (ΔIsc=3.6±0.4 vs. ΔIsc=0.7±0.2 µA/cm2, P< 0.05). SLC26A9-mediated currents were inhibited by niflumic acid (NFA; 100 µM; ΔIsc=1.7±0.6 µA/cm2) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 50 µM; ΔIsc=2.6±0.2 µA/cm2) (P<0.05). Under hypotonic conditions, basal Isc increased to 65.6±4.5 µA/cm2 in FRT-SLC and 45.4±3.3 µA/cm2 in FRT-CTL cell monolayers (P<0.01). IBMX/Forskolin-stimulated Cl- secretion in FRT-SLC (ΔIsc=5.6±0.8 µA/cm2) was significantly higher than in FRT-CTL cell monolayers (ΔIsc=1.3±0.3 µA/cm2) (P<0.05). In hypertonicity, FRT-SLC and FRT-CTL cell monolayers exhibited an elevated Isc of 69.4±5.6 µA/cm2 and 65.1±4.4 µA/cm2, respectively, albeit the effect of IBMX/Forskolin on Cl- secretion was significantly higher in FRT-SLC cell monolayers (FRT-SLC: ΔIsc=4.5±0.4 µA/cm2 vs. FRT-CTL: ΔIsc= 2.8±0.7 µA/cm2; P<0.05). Results, expressed as mean±SEM (n=7-18) were compared by ANOVA. We conclude that our new HA tagged-SLC26A9 overexpressing FRT epithelial cell line is a suitable in vitro model for further investigations on the regulation and pharmacology of the SLC26A9 Cl- channel. Our results indicate that activation of Cl- conductance in hypo- and hypertonic conditions in epithelial cells may require translocation of SLC26A9. Osmotic stress-sensing kinases such as WNK or SPAK/OSR1 complexes may be regulating this process.