Cochlear endolymph has highly positive potential of approximately ~+80 mV known as the endocochlear potential (EP). The EP is essential for hearing and maintained by K⁺ circulation from perilymph to endolymph through the cochlear lateral wall. Various K⁺ transport apparatuses such as the Na⁺,K⁺ ATPase, the Na⁺,K⁺,2Cl^-- cotransporter, and the K⁺ channels Kir4.1 and KCNQ1/KCNE1 are expressed in the lateral wall and known to play indispensable roles in cochlear K⁺ circulation. The gastric type of the H⁺,K⁺ ATPase was also shown expressed in the cochlear lateral wall, but its functional role has not been well studied. Here we examined the precise localization of H⁺,K⁺ ATPase in cochlea and its involvement in formation of EP. RT PCR analysis showed that the cochlea expressed mRNAs of gastric 1, but not colonic ₂, and subunits of H⁺,K⁺ ATPase. Immunolabeling of an antibody specific to the ₁ subunit was detected in type II, IV, and V fibrocytes distributed in the spiral ligament of the lateral wall and in the spiral limbus. Strong immunoreactivity was also found in the stria vascularis. Immunoelectron microscopic examination exhibited that the H⁺,K⁺-ATPase was localized exclusively at the basolateral site of strial marginal cells. Application of Sch28080, a specific inhibitor for gastric H⁺,K⁺ ATPase, to the spiral ligament as well as to the stria vascularis caused prominent reduction of EP. These results may imply that the H⁺,K⁺-ATPase in the cochlear lateral wall is crucial for K+ circulation wall and thus plays a critical role in generation of EP.