Ion homeostasis is a fundamental cellular process particularly important in excitable cell activities such as hearing. It relies on the Na(+)/K(+) ATPase (also referred to as the Na pump), which is composed of a catalytic α subunit and a β subunit required for its transport to the plasma membrane and for regulating its activity. We show that α and β subunits are expressed in Johnston's organ (JO), the Drosophila auditory organ. We knocked down expression of α subunits (ATPα and α-like) and β subunits (nrv1, nrv2, and nrv3) individually in JO with UAS/Gal4-mediated RNAi. ATPα shows elevated expression in the ablumenal membrane of scolopale cells, which enwrap JO neuronal dendrites in endolymph-like compartments. Knocking down ATPα, but not α-like, in the entire JO or only in scolopale cells using specific drivers, resulted in complete deafness. Among β subunits, nrv2 is expressed in scolopale cells and nrv3 in JO neurons. Knocking down nrv2 in scolopale cells blocked Nrv2 expression, reduced ATPα expression in the scolopale cells, and caused almost complete deafness. Furthermore, knockdown of either nrv2 or ATPα specifically in scolopale cells causes abnormal, electron-dense material accumulation in the scolopale space. Similarly, nrv3 functions in JO but not in scolopale cells, suggesting neuron specificity that parallels nrv2 scolopale cell-specific support of the catalytic ATPα. Our studies provide an amenable model to investigate generation of endolymph-like extracellular compartments.