In rat ileum and colon, apical membrane Cl(-)/HCO(3)(-) exchange and net Cl(-) absorption are stimulated by increases in Pco(2) or [HCO(3)(-)]. Because changes in Pco(2) stimulate colonic Na(+) absorption, in part, by modulating vesicular trafficking of the Na(+)/H(+) exchanger type 3 isoform to and from the apical membrane, we examined whether changes in Pco(2) affect net Cl(-) absorption by modulating vesicular trafficking of the Cl(-)/HCO(3)(-) exchanger anion exchanger (AE)1. Cl(-) transport across rat distal ileum and colon was 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 Pco(2) 70 mmHg than at Pco(2) 21 mmHg but was not affected by pH changes in the absence of CO(2). AE1 was internalized when Pco(2) was reduced and exocytosed when Pco(2) was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl(-) absorption caused by an increase in Pco(2). Increases in Pco(2) stimulated ileal Cl(-) absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by Pco(2). We conclude that CO(2) modulation of colonic, but not ileal, Cl(-) absorption involves effects on vesicular trafficking of AE1.