Glucose alters absorptive cell tight junction structure and, as deduced from an impedance analysis model, diminishes tight junction resistance in the small intestine (J.R. Pappenheimer, J. Membr. Biol. 100: 137-148, 1987; and J.L. Madara and J.R. Pappenheimer, J. Membr. Biol. 100: 149-164, 1987). Here we provide further evidence in support of this hypothesis using the conventional approach of analysis of mucosal sheets mounted in Ussing chambers. This approach offers advantages for investigating underlying mechanisms, including the effects of ions and inhibitors on the regulation of intercellular junctions by glucose. We show that phlorizin blocks a resistance decrease elicited by glucose and demonstrate that substitution of choline for sodium also prevents the response. The dilatations in absorptive cell tight junctions that accompany this glucose-elicited response are similarly prevented by phlorizin exposure or sodium substitution. The effects of phlorizin on junctional permeability can also be demonstrated in vivo. Phlorizin reduces the transjunctional flux of creatinine in glucose-perfused intestines of anesthetized animals, even when account is taken of the reduction of fluid absorption caused by phlorizin. Last, in vivo perfusion studies suggest that although, at 25 mM luminal glucose, virtually all glucose absorption is transcellular, at a luminal glucose concentration of 125 mM approximately 30% of glucose absorption occurs paracellularly because of solvent drag across tight junctions of altered permeability.(ABSTRACT TRUNCATED AT 250 WORDS)