The distribution of five Na,K-ATPase subunit isoforms (α1, α2, α3, β1 and β2) in rat cochlear and vestibular tissues was determined by immunocytochemical techniques using subunit isoform specific polyclonal antibodies. The expression of Na,K-ATPase α and β subunit isoforms varied among different cell regions of the inner ear. The α1 subunit isoform was more extensively distributed in all inner ear tissues than the α2 or α3 subunit isoforms. The β1 subunit isoform was distributed primarily in spiral ligament and inner hair cells of the cochlea, and in crista ampullaris and macula of the saccule. The β2, subunit isoform was most abundant in the stria vascularis, dark cells of the ampullae and utricle. The α1β1 subunit combination of Na,K-ATPase was most commonly found in the spiral ligament, while the α1β2 combination was most abundant in the stria vascularis. Similarly, α1β2 was confined more to the dark cells of the ampullae and utricle. The α3β1 suhunit combination of Na,K-ATPase was identified in the inner hair cells of the cochlea and the sensory regions of the vestibular end organs. These observations may reflect functional diversity of Na,K-ATPase in the individual inner ear regions and may provide insight into the differences between fluid and ion transport in the inner ear and that of other transporting tissues. Overall, the distribution pattern further indicates that the different isoform combinations have specific roles. © 1994.