We examined for vesicular trafficking of the Na⁺/H⁺ exchanger (NHE) in pH-stimulated ileal and CO₂-stimulated colonic Na⁺ absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at ×27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na⁺ absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na⁺ absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a PCO₂ value of 70 than 21 mmHg, was internalized when PCO₂ was reduced, and was exocytosed when PCO₂ was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na⁺ absorption but is important in CO₂-stimulated colonic Na⁺ absorption.