Background: Bicarbonate secretion protects the proximal duodenum against damage by gastric acid. Duodenocytes express CFTR as well as at least three members of the SLC26 family (Slc26a3, 6 and 9) in the apical membrane. Aim and Methods: To establish the importance of the different apical anion transporters during basal, acid- and forskolin (FSK)- stimulated duodenal HCO₃^- secretion in vivo, CFTR, Scl26a3, 6 and 9-deficient mice and WT littermates were anesthetized, the proximal duodenum was luminally perfused with saline, 10^-4 M FSK, or pH 2.2 for 5 min, and bicarbonate secretion was continuously titrated in the perfusate during controlled systemic acid/base parameters. Gene expression was assessed by villus and crypt cell laser dissection and qPCR. Results: Basal duodenal HCO₃^- secretion was slightly reduced in the Slc26a6 -/-, and more strongly reduced in the Slc26a3 -/-, Slc26a9, and CFTR-deficient mice compared to WT littermates. FSK-stimulated secretory response was normal in Slc26a6 -/-, Slc26a3 -/-, Slc26a9 - /- mice, and was virtually abolished in CFTR- deficient mice. Surprisingly, acid-activated HCO₃^- secretion was unaltered in the Slc26a6 -/- duodenum, but strongly reduced in Slc26a3 -/- and Slc26a9 -/- duodenum, and abolished in the CFTR-deficient duodenum. 20mM CFTR_(inh)-172 inhibited both acid- and FSK-stimulated HCO₃^- secretory response by approx. 50%. Laser dissection and immunohistochemistry revealed a villous- predominant expression of Slc26a6, Slc26a3 and a crypt-predominant expression of CFTR and Slc26a9. Conclusions: Genetic deletion of intestinal Slc26 anion transporters reveals their differential involvement in basal, acid- and FSK-stimulated HCO₃^- secretion in murine duodenum. Most likely, different signalling in acid- vs. FSK-stimulated HCO₃^- secretion explains this differential involvement, rather than the differential crypt-villus expression pattern.