It is known that the claudin family of tight junction proteins is critical in determining paracellular ionic permeability and selectivity. We have shown that loss of claudin 15 results in decreased luminal Na+ concentration and glucose malabsorption in the small intestine. To gain further insight into the relationship between intestinal Na+ metabolism and changes in nutrient absorption induced by the loss of claudin 15, we investigated the site of absorption of electrolytes and nutrients in claudin 15 knockout (cldn15KO) mice and compared this with wild-type mice under in vivo conditions. Mice were fed a powdered diet supplemented with 14C-polyethylene glycol (PEG) 4000 as a non-absorbable marker and 3H-Gly-Sar (non-hydrolyzable dipeptide) or glucose. Three hours after feeding, the small intestine was isolated and divided into six segments, the luminal contents collected for analysis of Na+, K+, and Cl- concentrations and the level of 14C-PEG4000. Na+, K+, and Cl- concentrations were determined using ion-selective electrodes. Glucose concentration was determined by colorimetry. Gastric emptying time, assessed by measuring 14C-PEG4000, was decreased in cldn15KO compared to wild-type mice. Total luminal contents in the small intestine were increased in cldn15KO mice and the retention time of digesta in the upper jejunum was increased approximately 3-fold compared with wild-type mice. Robust Na+ secretion and rate of absorption were observed in the upper jejunum in wild-type mice and this was attenuated in cldn15KO mice. The rate of K+ absorption was increased in cldn15KO mice in the lower ileum. Total luminal glucose and Gly-Sar were increased, while absorption rates of glucose and Gly-Sar were decreased, in the upper jejunum of cldn15KO mice. These results suggest that the sites of absorption and secretion of electrolytes and nutrients are changed by adaptation to the loss of claudin 15.