Bicarbonate transport across the duodenal epithelium establishes an alkaline mucus layer that protects the epithelium from gastric acid and provides an environment favorable to efficient nutrient digestion/absorption. The process of transepithelial HCO₃^– secretion involves the concerted activities of CFTR and apical membrane anion exchangers, including Slc4a9 (anion exchanger 4, AE4) and two members of the multifunctional anion exchanger family, Slc26a3 (down-regulated in adenoma, DRA) and Slc26a6 (putative anion transporter-1, PAT-1) (1,2). Studies of transepithelial HCO₃^– secretion using wild-type (WT) and CFTR(-) murine duodena have shown that basal secretion is reduced in the absence of CFTR and that the process is primarily mediated by Cl^–/HCO₃^– exchangers because secretion is abolished by removal of luminal Cl^–. During cAMP stimulation, secretion is apparently dependent on a CFTR-mediated HCO₃^– conductance because secretion is essentially unaffected by luminal Cl^– removal and abolished in the absence of CFTR. However, important exceptions to the dominance of a CFTR HCO₃^– conductance during cAMP-stimulated secretion have been identified whereby ~50% of cAMP-stimulated HCO₃^– secretion is not associated with the short-circuit current (Isc), i.e. electroneutral (3). To further investigate the interactions of CFTR and the anion exchangers in native intestine, we measured intracellular pH in the villous epithelium of murine duodenum placed in a horizontal Ussing chamber. Contributions of the specific anion exchangers were investigated using mice with gene-targeted deletions of AE4, DRA, PAT-1 and/or CFTR. Upper villous epithelium. Earlier studies proposed that Cl^– channels enhance the activity of anion exchangers by providing a “leak” pathway that sustains the Cl_out:Cl_in concentration gradient. More recent studies suggest that CFTR directly associates with anion exchange proteins to increase their activity (4). In studies of the upper villous epithelium, we found that the basal rate of Cl^–/HCO₃^– exchange was significantly reduced in the CFTR(-) duodenum. Ion substitution revealed that greater transport rates in WT duodenum were detected only when the anion exchanged for HCO₃^– was CFTR-permeable. Further, glybenclamide block of CFTR reduced the rate of Cl^–/HCO₃^– exchange in WT to a level equivalent with CFTR(-) epithelium. Thus, the anion channel activity of CFTR rather than its mere presence in the apical membrane is required to promote basal Cl^–/HCO₃^– exchange. Studies of anion exchanger knockout mice demonstrated that Cl^–/HCO₃^– exchange was unchanged in the AE4(-) duodenum. In contrast, anion exchange was decreased by ~20% in the DRA(-) and ~80% in the PAT-1(-) duodenum, respectively. It was concluded that CFTR indirectly facilitates the activity of PAT-1 in the villous epithelium by providing a Cl^– leak pathway. DRA may not participate due to coupling with the Na⁺/H⁺ exchanger, NHE3, for NaCl absorption. Lower villous epithelium. Studies comparing WT and knockout duodena revealed that Cl^–/HCO₃^– exchange in the lower villous epithelium was not altered in the PAT-1(-) mice, but completely abolished in DRA(-) mice. Paradoxically, removal of luminal Cl^– in the DRA(-) lower villus caused cell acidification, which was CFTR-dependent because luminal Cl^– removal did not alter the pH_i in the DRA(-)/CFTR(-) duodenum. The outcome of these experiments is consistent with a report that the HCO₃^– permeability of CFTR is increased by removal of extracellular Cl^– (5). Contributions of PAT-1 and DRA to transepithelial HCO₃^– secretion. pH stat measurements of PAT-1(-) duodenum revealed that PAT-1 only contributes to basal (~25%) but not cAMP-stimulated HCO₃^– secretion. In contrast, DRA was responsible for ~50% of both basal and cAMP-stimulated HCO₃^– secretion in physiological (i.e. Cl^– containing) medium. The cAMP-stimulated Isc response was unchanged in the DRA(-) intestine, indicating normal CFTR activity. The remaining component of stimulated HCO₃^– secretion can be ascribed to a CFTR-mediated HCO₃^– conductance, which would comprise ~15% of the stimulated Isc, i.e. approximating the HCO₃^–:Cl^– permeability ratio of CFTR. Interestingly, cAMP-stimulated HCO₃^– secretion was normalized in the DRA(-) duodenum by luminal Cl^– removal, again suggesting that the CFTR-mediated HCO₃^– conductance is enhanced in the absence of extracellular Cl^–. In summary, in the upper villous epithelium where expression is low, CFTR plays a passive role as a Cl^– leak pathway that facilitates the activity of PAT-1. In the lower villous epithelium, CFTR’s association confers the ability for cAMP stimulation to DRA. Understanding the roles that PAT-1 and DRA play in duodenal HCO₃^– secretion may lead to therapies normalizing juxtamucosal pH to correct deficiencies of nutrient digestion and absorption in the CF intestine.