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). Particular interest focuses on the role of CFTR in HCO₃^– secretion, the loss of which is thought to underlie much of the pathology of cystic fibrosis. The aim of these studies was to characterise the acid/base transporters that are expressed in the apical membrane of Calu-3 cells grown as polarised monolayers on Transwell polyester filters (Corning). Filters were superfused bilaterally with Hepes- or HCO₃^–-buffered solutions and maintained at 37°C on the stage of an inverted fluorescence microscope. Cells were loaded with the pH-sensitive fluoroprobe BCECF and intracellular pH (pH_i) was recorded by standard microfluorometric techniques. The recovery of pH_i from an acid load induced by brief bilateral exposure to NH₄⁺ was entirely dependent on extracellular Na⁺. In HCO₃^–-free conditions there was a significant recovery of pH_i when Na⁺ was restored to the apical bath in the absence of basolateral Na⁺. This was blocked by apical EIPA (30 μM) and therefore attributed to apical expression of an Na⁺/H⁺ exchanger, most probably NHE2 or -3. When repeated in the presence of HCO₃^–, there was no evidence for the involvement of any additional Na⁺-dependent processes at the apical membrane. Substitution of Cl^– by gluconate on the apical side of the Calu-3 cells led to a small increase in pH_i (0.06 ± 0.02, mean ± s.e.m., n = 8, P < 0.05 by paired t test). However, following combined stimulation with forskolin (3 μM) and IBMX (100 μM) to elevate intracellular cAMP, apical Cl^– substitution resulted in a large and rapid increase in pH_i 0.37 ± 0.03, n = 9, P < 0.001). Although this alkalinization might normally be attributed to Cl^–/HCO₃^– exchange across the apical membrane, it was relatively insensitive to the non-specific anion exchange inhibitor DIDS (28% inhibition with 100 μM and 57% with 500 μM). In contrast, it could be totally abolished by the specific CFTR channel blocker CFTRinh-172 (79% inhibition with 5 μM and 95% with 10 μM). This suggests either that the alkalinization is due to HCO₃^– entry through the CFTR channel, which would require a reversal of the apical membrane potential, or that the depolarization resulting from Cl^– efflux through CFTR alters the driving forces for other electrogenic acid/base transporters.