In rat ileum and colon, apical membrane Cl^-/HCO₃^- exchange and net Cl^- absorption are stimulated by increases in Pco₂ or [HCO₃^-]. Since changes in Pco₂ stimulate colonic Na⁺ absorption in part by modulating vesicular trafficking of the NHE3 isoform to and from the apical membrane, we examined whether changes in Pco₂ affect net Cl^- absorption by modulating vesicular trafficking of the Cl^-/HCO₃^- exchanger AE1. Cl^- transport across rat distal ileum and colon were measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at Pco₂ 70 mmHg than at Pco₂ 21 mmHg but was not affected by pH changes in the absence of CO₂. AE1 was internalized when Pco₂ was reduced, exocytosed when Pco₂ was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl^- absorption caused by an increase in Pco₂. Increases in Pco₂ stimulated ileal Cl^- absorption but wortmannin was without affect. Ileal epithelial apical membrane AE1 content was not affected by Pco₂. We conclude that CO₂-modulation of colonic, but not ileal, Cl^- absorption involves effects on vesicular trafficking of AE1.