Backround: The molecular mechanisms for nutrient- coupled electrolyte and fluid absorption are incompletely understood. Objective and methods: In order elucidate the mechanisms of nutrient- induced salt absorption, we measured the in vivo jejunal fluid absorption, and in vitro enterocyte pHi changes, during exposure to glucose (taken up via SGLT1-mediated Na+-glucose cotransport), and fructose (via GLUT5-mediated facilitated diffusion) in mice defi-cient for the apical Na+/H+ exchanger NHE3, and the apical Cl-/HCO3- exchangers Slc26a3 and Slc26a6, and WT littermates. Results: Replacement of 20 mM Mannitol by 20 mM of glucose stimulated jejunal fluid absorption in vivo. The application of both nutrients caused a significant decline in enterocyte pHi in intact jejunal villi, which was significantly stronger in the absence of NHE3 or SLC26a6, reflecting the activation of apical ion transporters through glucose-induced enterocyte pHi decrease. This pHi-decline occurred also with a non-metabolizable substrate for SGLT1, and was reduced by the anion channel blocker NPPB or the K+ blocker TEA, and therefore likely due to the activation of volume- regulatory anion channels during nutrient uptake. Fructose-mediated fluid absorption in vivo was abolished in the NHE3-deficient jejunum and significantly reduced in the Slc26a6- deficient jejunum, whereas glucose-induced fluid absorption was normal. Slc26a3-deficient jejunum had mark-edly reduced basal fluid absorptive rates but the glucose- or fructose-induced increase was normal. Conclusions: Small intestinal absorption of sugars or results in enterocyte acidifica-tion, most likely due to regulatory volume decrease, which in turn stimulates apical Na+/H+ and Cl-/HCO3- exchangers and results in salt and fluid absorption. While Na+-nutrient co-transport systems activate the same apical Na+/H+ and Cl- /HCO3- exchangers than non-Na+ coupled nutrient uptake mechanisms, they are less dependent on them for stimulating fluid absorption.