Background: HCO₃^– 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. Mice were anesthetized by a spontaneous inhalation of isoflurane (Forene, Abbott Scandinavia, Kista, Sweden). The inhalation gas contained a mixture of ~20-30% oxygen, ~70-80% air, and 2.0 +/- 0.2% isoflurane with the use of an isoflurane pump (Univentor 400 Anaesthesia Unit, AgnTho, Lidingo¨, Sweden) and was administered continuously through a breathing mask. The depth of the anaesthesia was tested by probing the pedal reflex. CFTR-, Scl26a3, 6 and 9-deficient mice and WT litter-mates were anesthetized, the proximal duodenum was luminally perfused with saline, 10^-4 M FSK, or pH 2.2 for 5 min, and HCO₃^– was continuously titrated in the perfusate during controlled systemic acid/base parameters. The relative crypt and villous expression of the trans-porters was measured by immunohistochemistry, laser dissection and qPCR. Results: Basal duodenal HCO₃^– secretion was slightly reduced in the Slc26a6^-/-, and more strongly reduced in the Slc26a3^-/-, Slc26a9^-/-, and in CFTR-deficient mice compared to WT littermates. FSK-stimulated secretory response was normal in Slc26a3^-/-, Slc26a6^-/-, 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. Laser dissection and immunohistochemistry revealed a villous-predominant expression of Slc26a6, Slc26a3 and NBCn1 and a crypt predominant expression of CFTR and Slc26a9. Conclusions: Genetic deletion of intestinal Slc26 anion transporters reveals their differential involvement in basal, acid-stimulated 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.