Tight junctions (TJs) create ion-selective paracellular permeability barriers between extracellular compartments. In the organ of Corti of the inner ear, TJs of the reticular lamina separate K(+)-rich endolymph and Na(+)-rich perilymph. In humans, mutations of the gene encoding claudin 14 TJ protein cause profound deafness but the underlying pathogenesis is unknown. To explore the role of claudin 14 in the inner ear and in other tissues we created a mouse model by a targeted deletion of Cldn14. In the targeted allele a lacZ cassette is expressed under the Cldn14 promoter. In Cldn14-lacZ heterozygous mice beta-galactosidase activity was detected in cochlear inner and outer hair cells and supporting cells, in the collecting ducts of the kidney, and around the lobules of the liver. Cldn14-null mice have a normal endocochlear potential but are deaf due to rapid degeneration of cochlear outer hair cells, followed by slower degeneration of the inner hair cells, during the first 3 weeks of life. Monolayers of MDCK cells expressing claudin 14 show a 6-fold increase in the transepithelial electrical resistance by decreasing paracellular permeability for cations. In wild type mice, claudin 14 was immunolocalized at hair cell and supporting cell TJs. Our data suggest that the TJ complex at the apex of the reticular lamina requires claudin 14 as a cation-restrictive barrier to maintain the proper ionic composition of the fluid surrounding the basolateral surface of outer hair cells.