Background and aim: The jejunum is known to be capable of absorbing large amounts of fluid and HCO3- , but the molecular mechanisms are not clear. We speculated that the large amounts of luminal CO2 and HCO3- generated in the upper jejunum by mixture of HCO3–rich pancreatic juice and H+-rich gastric juice, as well as the jejunal enterocyte proton load generated by H+-coupled dipeptide and aminoacid absorption, are major stimulants of jejunal fluid and Na+HCO3- absorption, and that both the apical Na+/H+ exchanger NHE3 and the multi anion transporter Slc26a6 (PAT-1) may be involved in dipeptide- and CO2/HCO3- induced jejunal fluid and Na+HCO3- absorption. Methods: Two photon fluorometry was applied to measure intracellular pH (pHi) in SNARF1-loaded villous enterocytes of exteriorized jejunum in anesthetized C57BL6 background mice. Jejunal fluid absorption was assessed by single pass perfusion of jejunal segments in anesthetized acid/base-status controlled mice with prewarmed, isotonic solutions of different ionic compositions but constant pH of 7.4; volume and pH of the inflowing and outflowing solution was measured. Results: WT jejunum absorbed fluid and acidified the lumen. Slc26a3 (DRA)-deficient jejunum absorbed less fluid than WT, and acidified the lumen more strongly, consistent with the action of DRA as a Cl-/HCO3- exchanger. Slc26a6 (PAT-1)-deficient jejunum also absorbed less fluid, but resulted in less luminal acidification. NHE3-deficient jejunum also absorbed less fluid, and markedly alkalinized the lumen. Switching the luminal perfusate from isotonic NaCl/O2 gassed to isotonic NaCl/HCO3-, 5%CO2/95%O2 gassed solution, pH 7.4, resulted in stable decrease of villous enterocyte pHi by approx. 0.2 pH units, and a marked stimulation fluid absorption in a PAT-1 – and NHE3- but not DRA-dependent fashion. Switching the solution from CO2/HCO3- buffered Mannitol-containing saline to an isotonic solution where the mannitol was preplaced by 20 mM of the dipeptide GlySar further acidified enterocyte pH and resulted in a further increase of fluid absorption that was dependent on PAT-1 as well as NHE3. Conclusions: The results suggest that luminal CO2/HCO3-, as well as luminal dipeptides, strongly stimulates of jejunal fluid absorption, which occurs to a large part percentage secondary to Na+HCO3- absorption. NHE3 and Slc26a6 (PAT-1) are required for this CO2/HCO3– and dipeptide-augmented Na+HCO3- absorption, and Slc26a6 (PAT-1) may operate in a 2 HCO3-/1 Cl- mode under those circumstances. The results demonstrate an important role of Slc26a6 (PAT-1) in jejunal fluid and HCO3- absorption.