In cystic fibrosis (CF) mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel leads to reduced airway Cl^– and HCO₃^– -dependent fluid secretion which impairs airway defence mechanisms and predisposes the lungs to bacterial infection (1). CFTR is regarded as the sole mediator of apical Cl^– and HCO₃^– secretion in airway epithelial cells. However, the discovery of SLC26 Cl^–/HCO₃^– anion exchange (AE) activity linked to the expression of CFTR in tracheal and pancreatic duct epithelial cells (2,3) as well as the demonstration of a functional interaction between SLC26 exchangers and CFTR (4), has led us to re-evaulate this hypothesis. The aim of this work was to investigate the role of SLC26 exchangers in HCO₃^– and fluid secretion from human airway cells. AE activity was assessed by real time measurements of intracellular pH (pH_i), using the pH-sensitive dye BCECF-AM, from Calu-3 cells grown as monolayers on semi-permeable supports (3). Calu-3 cells are used as a model of the serous cells of human airway submucosal glands and display CFTR-dependent HCO₃^– secretion (5). Under resting conditions apical Cl^– removal had no effect on pH_i, but caused an alkalinisation of 0.60±0.07 (mean ± s.e.m.;P<0.001 paired t-test, n=8) after 5 mins of 5μM forskolin stimulation. A similar alkalinisation was observed following expoure to 10μM adenosine (0.53±0.02, n=4) and 300 nM VIP (0.60±0.04, n=7). The apical AE activity was insensitive to H2DIDs, but blocked by the CFTR inhibitor GlyH-101 (10µM) and the PKA inhibitor H-89 (50µM). AE activity was also reduced by 50 % in Calu-3 cells where CFTR content was knocked down by 95%. The re-acidification in pH_i following restoration of apical Cl^– was mimicked by other monovalent anions including iodide, bromide, nitrate, formate and thiocyanate. However, apical AE activity was not supported by divalent anions such as oxalate or sulphate. The profile of this apical Cl^–/HCO₃^– exchanger is consistent with Pendrin (SLC26A4), which quantitative RT-PCR and immunohistochemical analysis showed to be expressed in Calu-3 cells. Fluid secretion studies showed that forskolin enhanced secretion nearly fourfold (20.0±1.0 to 73.7±3.0 µl/cm²/24hrs, n=4), which was significantly reduced by GlyH-101 (~60%) and much lower in the CFTR knock down Calu-3 cells (~58% reduction). The forskolin stimulated secretion was more alkaline than the control secretion (7.77±0.02 viz 7.38±0.01, n=4) and had a lower Cl^– concentration (107±4mM viz 122±3, n=4), data consistent with forskolin stimulating CFTR-dependent bicarbonate secretion via a Cl^–/HCO₃^– exchange mechanism. We propose that impaired Cl^– transport, as well as defective regulation of apical anion exchangers, underlies the reduced, acidic, fluid secretion in CF airways.