Background and aim: Duodenal mucosal bicarbonate secretion (DBS) is considered an important protective mechanism against acid from the stomach, and is defective in duodenal ulcer disease. Carbonic anhydrase (CA) is strongly expressed in the proximal small intestine and has been implicated in a variety of physiological functions including enterocyte bicarbonate supply for secretion and the sensing of luminal acid. The aim of the present study was to investigate the role of the intracellular CAII isoform involvement in acid-induced murine DBS in vivo. Methods: A ~10-mm segment of the proximal duodenum with intact blood supply was perfused under different experimental conditions and DBS was titrated by pH-stat. Two-photon confocal microscopy using the pH-sensitive dye SNARF-1F were used to assess duodenocyte pHi in vivo. Results: The duodenal bicarbonate secretory response to acid was abolished in CAII-deficient mice, but normal to forskolin- or 16,16-dimethyl prostaglandin E2 stimulation. Complete inhibition of CAs by luminal methazolamide and intravenous acetazolamide completely inhibited the response to acid, but did not alter the response to forskolin. While duodenocytes acidified upon luminal perfusion with acid, no significant pHi changed occurred in CAII-deficient duodenum. Conclusions: Our results show that CAII is essential for duodenocyte acidification after exposing the duodenal lumen to a low pH and for the acid-mediated bicarbonate secretory response, but is not critical for the generation of the secreted bicarbonate ions.