The Drosophila genome contains at least three loci for the Na,K-ATPase beta-subunit; however, only the protein products of nrv1 and nrv2 have been characterized hitherto. Here, we provide evidence that nrv3 also encodes for a functional Na,K-ATPase beta-subunit, as its protein product co-precipitates with the Na,K-ATPase alpha-subunit. Nrv3 expression in adult flies is restricted to the nervous system in which Nrv3 is enriched in selective types of sensory cells. Because Nrv3 expression is especially prominent in the compound eye, we have analyzed the subcellular and developmental distribution of Nrv3 within the visual cells and related this distribution to those of the alpha-subunit and of the beta-subunits Nrv1 and Nrv2. Prospective visual cells express Nrv2 in the third larval instar stage and during the first half of pupal development. During the last third of pupal life, Nrv3 gradually replaces Nrv2 as the Na,K-ATPase beta-subunit in the photoreceptor cells. Adult photoreceptors express Nrv3 as their major beta-subunit; the visual cells R1-R6 co-express Nrv2 at a low level, whereas R7 and R8 co-express Nrv1. Notably, beta-subunits do not co-distribute exactly with the alpha-subunit at some developmental stages, supporting the concept that the alpha-subunit and beta-subunit can exist in the plasma membrane without being engaged in alpha/beta heterodimers. The non-visual cells within the compound eye express almost exclusively Nrv2, which segregates together with the alpha-subunit to septate junctions throughout development.