The majority of dietary amino acids are absorbed via the H(+)-di-/tripeptide transporter Pept1 of the small intestine. Proton influx via Pept1 requires maintenance of intracellular pH (pH(i)) to sustain the driving force for peptide absorption. The apical membrane Na(+)/H(+) exchanger Nhe3 plays a major role in minimizing epithelial acidification during H(+)-di-/tripeptide absorption. However, the contributions of HCO(3)(-)-dependent transporters to this process have not been elucidated. In this study, we investigate the role of putative anion transporter-1 (Pat-1), an apical membrane anion exchanger, in epithelial pH(i) regulation during H(+)-peptide absorption. Using wild-type (WT) and Pat-1(-) mice, Ussing chambers were employed to measure the short-circuit current (I(sc)) associated with Pept1-mediated glycyl-sarcosine (Gly-Sar) absorption. Microfluorometry was used to measure pH(i) and Cl(-)/HCO(3)(-) exchange in the upper villous epithelium. In CO(2)/HCO(3)(-)-buffered Ringers, WT small intestine showed significant Gly-Sar-induced I(sc) and efficient pH(i) regulation during pharmacological inhibition of Nhe3 activity. In contrast, epithelial acidification and reduced I(sc) response to Gly-Sar exposure occurred during pharmacological inhibition of Cl(-)/HCO(3)(-) exchange and in the Pat-1(-) intestine. Pat-1 interacts with carbonic anhydrase II (CAII), and studies using CAII(-) intestine or the pharmacological inhibitor methazolamide on WT intestine resulted in increased epithelial acidification during Gly-Sar exposure. Increased epithelial acidification during Gly-Sar exposure also occurred in WT intestine during inhibition of luminal extracellular CA activity. Measurement of Cl(-)/HCO(3)(-) exchange in the presence of Gly-Sar revealed an increased rate of Cl(-)(OUT)/HCO(3)(-)(IN) exchange that was both Pat-1 dependent and CA dependent. In conclusion, Pat-1 Cl(-)/HCO(3)(-) exchange contributes to pH(i) regulation in the villous epithelium during H(+)-dipeptide absorption, possibly by providing a HCO(3)(-) import pathway.