The Calu-3 cell line, derived from human airway submucosal glands, expresses high levels of CFTR and is a good model for investigating Cl⁻ and HCO₃⁻ secretion in an epithelium that is adversely affected by cystic fibrosis (Hug et al., 2003). The aim of these studies was to determine whether HCO₃⁻ secretion across the apical membrane is mediated mainly by CFTR or by a Cl⁻/HCO₃⁻ exchanger of the SLC26 family, as is the case in several other epithelia. Calu-3 cells were grown as polarised monolayers on Transwell polyester filters (Corning), superfused bilaterally with HCO₃⁻-buffered solutions and maintained at 37°C on the stage of an inverted fluorescence microscope. Intracellular pH (pH_i) was measured with BCECF using standard microfluorometric techniques. Substitution of Cl⁻ with gluconate in the apical bath led to a marked alkalinization (to pH 7.83 ± 0.14, mean ± s.e.m., n=13) in 10 µM forskolin-stimulated Calu-3 cells, suggestive of Cl⁻/HCO₃⁻ exchange. Unexpectedly, the alkalinization and subsequent recovery of pH_i were unaffected by the Cl⁻/HCO₃⁻ exchanger inhibitor DIDS (100 µM) but were markedly inhibited by the CFTR channel blocker CFTR_inh-172 (5 µM). This suggested that the alkalinization might be due to HCO₃⁻ entry through the CFTR channel rather than via a Cl⁻/HCO₃⁻ exchanger. In support of this interpretation, quantitative RT-PCR measurements indicated only low levels of SLC26 exchanger expression in Calu-3 cells (A3 <0.1%, A4 <0.1%, A6 10.4 ± 3.3% relative to CFTR, n=6) while expression of the basolateral anion exchanger AE2 was much greater (184 ± 36% relative to CFTR). However, for pH_i to rise to 7.8 through electrodiffusive entry of HCO₃⁻ via CFTR, the membrane potential would have to depolarize and reverse to a value of approximately +30 mV as a result of Cl⁻ efflux. Perforated patch-clamp recordings using gramicidin D confirmed that such large changes in membrane potential (to +27.5 ± 5.0 mV, n=5) did indeed occur in forskolin-stimulated Calu-3 cells exposed to zero extracellular Cl⁻. We therefore conclude that, unlike other HCO₃⁻-secreting epithelia, such as the duodenum and pancreatic duct, Calu-3 airway cells secrete HCO₃⁻ predominantly via CFTR rather than an SLC26 anion exchanger.