Villi of the proximal duodenum are situated for direct exposure to gastric acid chyme. However, little is known about active bicarbonate secretion across villi that maintains the protective alkaline mucus barrier, a process that may be compromised in cystic fibrosis (CF), i.e., in the absence of a functional CF transmembrane conductance regulator (CFTR) anion channel. We investigated Cl⁻/HCO₃⁻ exchange activity across the apical membrane of epithelial cells located at the midregion of villi in intact duodenal mucosa from wild-type (WT) and CF mice using the pH-sensitive dye BCECF. Under basal conditions, the Cl⁻/HCO₃⁻ exchange rate was reduced by ∼35% in CF compared with WT villous epithelium. Cl⁻/HCO₃⁻ exchange in WT and CF villi responded similarly to inhibitors of anion exchange, and membrane depolarization enhanced rates of Cl⁻_out/HCO₃⁻_in exchange in both epithelia. In anion substitution studies, anion_in/HCO₃⁻_out exchange rates were greater in WT epithelium using Cl⁻ or NO₃⁻, but decreased to the level of the CF epithelium using the CFTR-impermeant anion, SO₄²⁻. Similarly, treatment of WT epithelium with the CFTR-selective blocker glybenclamide decreased the Cl⁻/HCO₃⁻ exchange rate to the level of CF epithelium. The mRNA expression of Slc26a3 (downregulated in adenoma) and Slc26a6 (putative anion exchanger-1) was similar between WT and CF duodena. From these studies of murine duodenum, we conclude 1) characteristics of Cl⁻/HCO₃⁻ exchange in the villous epithelium are most consistent with Slc26a6 activity, and 2) Cl⁻ channel activity of CFTR facilitates apical membrane Cl⁻_in/HCO₃⁻_out exchange by providing a Cl⁻ “leak” under basal conditions.