PAT1 (SLC26A6) and NHE3 are located on the apical membrane of small intestinal villi and mediate chloride/bicarbonate exchange and Na+/H+ exchange, respectively. To ascertain the role of Slc26a6 and NHE3 in jejunal sodium and chloride absorption, muscle-stripped jejuna from Slc26a6 and NHE3 +/+ and -/- mice were mounted in Ussing chambers, and electrical parameters, and 36Cl- and 22Na+ fluxes were measured. In the basal state, net Cl- and Na+ fluxes were absorptive in both Slc26a6 -/- and +/+ jejuni, but significantly decreased in -/- animals. Upon forskolin addition, net Cl- flux became secretory in both Slc26a6 -/- and +/+ jejuni. When luminal glucose was added to activate Naglucose cotransport, concomitant Cl- absorption was significantly reduced in Slc26a6-/- jejuni. Identical experiments in NHE3-deficient jejuni showed reduced Na+ and Cl- absorption in the basal state, and the lack of NHE3, but not of SLC26A6, rendered Na+ as well as Cl- absorption unresponsive to inhibition by cAMP. On the other hand, glucose- mediated Na+ and Cl- absorption was normal in NHE3-deficient mice. The data strongly suggests two previously undiscovered functions for Slc26a6: It acts in concert with NHE3 in electroneutral salt absorption in the small intestine and also serves to absorb Cl- during glucose-driven salt absorption.