Claudins are cell adhesion molecules working at tight junctions (TJs) that are directly involved in compartmentalization in multicellular organisms. The cochlea includes a rather peculiar compartment filled with endolymph. This compartment is characterized by high K⁺ concentration (∼150 mM) and a positive endocochlear potential (∼90 mV; EP), both indispensable conditions for cochlear hair cells to transduce acoustic stimuli to electrical signals. These conditions are thought to be generated by the stria vascularis, which is adjacent to the endolymph compartment. The stria vascularis itself constitutes an isolated compartment delineated by two epithelial barriers, marginal and basal cell layers. Because TJs of basal cells are primarily composed of claudin-11, claudin-11-deficient (Cld11^-/-) mice were generated with an expectation that the compartmentalization in stria vascularis in these mice would be affected. Auditory brainstem response measurements revealed that Cld11^-/- mice suffered from deafness; although no obvious gross morphological malformations were detected in Cld11^-/- cochlea, freeze-fracture replica electron microscopy showed that TJs disappeared from basal cells of the stria vascularis. In good agreement with this, tracer experiments showed that the basal cell barrier was destroyed without affecting the marginal cell barrier. Importantly, in the endolymph compartment of Cld11^-/- cochlea, the K⁺ concentration was maintained around the normal level (∼150 mM), whereas the EP was suppressed down to ∼30 mV. These findings indicated that the establishment of the stria vascularis compartment, especially the basal cell barrier, is indispensable for hearing ability through the generation/maintenance of EP but not of a high K⁺ concentration in the endolymph.