Water is important for all living things. In the intestinal tract, more than 10 L of water is absorbed per day, including water that is ingested and secreted into the intestinal lumen. The pathway of water transport in the intestinal epithelium is thought to be mediated by both transcellular and paracellular routes. Permselectivity of the paracellular pathway is characterized by tight junctions, which are composed of many proteins (1). It has been shown that claudin family proteins, which are mainly expressed in bicellular tight junctions, are important to permselectivity (2). However, in claudin knockout (KO) mice, changes in water absorption in the intestine were not observed, suggesting another mechanism is involved (3). Recently it was shown that knock out of ILDR1, a tricellular tight junction protein, results in water transport abnormalities in the kidney (4). ILDR1 KO mice had impaired urine concentration (390 vs 1460 mOsm/Kg H2O), polydipsia (34 mL H2O/day) and polyuria (7.8 mL urine/day)(4). Since ILDR1 KO mice have impaired water transport in the kidney, the goal of this research was to examine the effect of ILDR1 knockout on the paracellular transport of water and electrolytes in the large intestine. ILDR1 KO mice (Ildr1tm1(KOMP)Wtsi; Wellcome Trust Sanger Institute Mouse Genetics Project) and their controls (age- and sex-matched C57Bl/6JJ mice) were put into metabolic cages for 72 hours and daily food and water intake was recorded and urine and feces were weighed and analyzed. To examine the effect of ILDR1 on paracellular transport, 22Na and 3H-mannitol fluxes were measured with the Ussing chamber method. Urine for osmolarity quantification was extracted directly from the bladder. All animal experiments were approved by the University of Shizuoka Animal Care and Use Committee (Permit #175179) and carried out in accordance with the rules for animal research at the University of Shizuoka. Anaesthesia (30 µg/mL medetomidine, 0.4 mg/mL midazolam, and 0.5 mg/mL butorphanol) was administered by intraperitoneal injection (10 µL/g body weight). There was no significant difference between wild type and ILDR1 KO mice in urine and feces Na+ and K+ concentration. Water and food intake as well as urine and fecal output did not differ in ILDR1 KO mice and controls. Large intestinal luminal Na+ and K+ concentration did not vary between wild type and ILDR1 KO mice. In addition, there was no difference in 22Na and 3H-mannitol flux in wild type and KO mice and conductance was also unchanged. Since there was no difference in urine osmolarity with free access to food and water, wild type and ILDR1 KO mice were subjected to 24 hour water restriction. ILDR1 KO mice were able to concentrate urine to a lesser extent than wild type (2620.5 ± 167.3 vs 3673.5 ± 87.7 mOsm, n=4, p=0.0014, independent t-test). Based on these results, ILDR1 knockout does not significantly impact water transport in the large intestine or kidney.