The sodium pump Na+/K+-ATPase, expressed in virtually all cells of higher organisms, is involved in establishing a resting membrane potential and in creating a sodium gradient to facilitate a number of membrane-associated transport activities. Na+/K+-ATPase is an oligomer of α, β, and γ subunits. Four unique genes encode each of the α and β subunits. In dental enamel cells, the spatiotemporal expression of Na+/K+-ATPase is poorly characterized. Using the rat incisor as a model, this study provides a comprehensive expression profile of all four α and all four β Na+/K+-ATPase subunits throughout all stages of amelogenesis. Real-time PCR, western blot analysis, and immunolocalization revealed that α1, β1, and β3 are expressed in the enamel organ and that all three are most highly expressed during late-maturation-stage amelogenesis. Expression of β3 was significantly higher than expression of β1, suggesting that the dominant Na+/K+-ATPase consists of an α1β3 dimer. Localization of α1, β1, and β3 subunits in ameloblasts was primarily to the cytoplasm and occasionally along the basolateral membranes. Weaker expression was also noted in papillary layer cells during early maturation. Our data support that Na+/K+-ATPase is functional in maturation-stage ameloblasts.