Background and Aims: Slc26a3 and a6 are members of a multi-anion transporting family and both mediate Cl-/HCO3- exchange. Both play important roles in intestinal HCO3- secretion and electroneutral salt absorption, but only Slc26a6 can either export or import HCO3- depending on pHi. In addition, only Scl26a6 has a carbonic anhydrase (CA) binding motif. CAs catalyze the reversible conversion of CO2 to HCO3-, and some anion transporters form transport metabolon with CAs, to maximize the coupled catalytic/transport flux. The aim of this study was to find evidence for functional coupling and co-localization of Slc26a3 and a6 with CAII in vivo. Methods and Results: CAII was found to co-localize with Slc26a6 in the apical membrane of villous enterocytes by immunohistochemistry in WT and Slc26a3 KO mice, while it had a cytoplasmic distribution in Slc26a6-deficient villi. Further, CAII could be co-immunoprecipitated with Slc26a6 from the native intestine. The amount of total CAII was reduced in Slc26a6-deficient duodenum as shown by western blot. The proximal duodenum of isoflurane-anesthetized (2% in 10-15% O2 and 85-90% air) Slc26a3, a6, CAII KO, or double KO and WT mice was perfused in situ, and basal and PGE2-stimulated HCO3- secretion was determined by back titration. Deletion of Slc26a6 slightly reduced both the basal rate and more so the PGE2-stimulated response, and this reduction was dramatically augmented by additional CAII ablation, which, by itself, did not have a significant effect. This suggests that the tethering of CAII to the apical membrane by Slc26a6 does not only affect transport of HCO3- via Slc26a6, but also via other HCO3- transporters such as CFTR or Slc26a3. Similar results were obtained by luminal application of the carbonic anhydrase inhibitor methazolamide (MTZ). Slc26a3 KO duodenum displayed reduced basal HCO3- secretory rate, and this difference was abolished by MTZ application. Slc26a3 KO mice had normal HCO3- response to PGE2 stimulation. Conclusion: The anion exchanger Slc26a6, which is strongly expressed in the brush border membrane of the upper intestine, serves as an apical membrane anchor for CAII. This appears to not only serve for HCO3- transport via Slc26a6 but also via other apical membrane HCO3- transporters Functional augmentation of Slc26a6 by CAII during HCO3- secretion in the murine duodenum in vivo may explain in part the strong dependency of Slc26a6 HCO3- export function on the blood acid/base parameters.