Basal HCO₃⁻ secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl⁻/HCO₃⁻ exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl⁻/HCO₃⁻ exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl⁻/HCO₃⁻ exchange activity was reduced by 65–80% in the PAT-1(−) duodenum, 30–40% in the DRA(−) duodenum, and <5% in the AE4(−) duodenum compared with the WT duodenum. SO₄²⁻/HCO₃⁻ exchange was eliminated in the PAT-1(−) duodenum but was not affected in the DRA(−) and AE4(−) duodenum relative to the WT duodenum. Intracellular pH (pH_i) was reduced in the PAT-1(−) villous epithelium but increased to WT levels in the absence of CO₂/HCO₃⁻ or during methazolamide treatment. Further experiments under physiological conditions indicated active pH_i compensation in the PAT-1(−) villous epithelium by combined activities of Na⁺/H⁺ exchanger 1 and Cl⁻-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl⁻/HCO₃⁻ and SO₄²⁻/HCO₃⁻ exchange across the apical membrane and 2) PAT-1 plays a role in pH_i regulation in the upper villous epithelium of the murine duodenum.