The expression pattern of the alpha and beta isoforms and the gamma subunit of the Na,K-ATPase was investigated during in vitro induction of pluripotent murine embryonic stem (ES) cells into neuronal cells. alpha1 protein was expressed in undifferentiated ES (UES) cells and throughout all stages studied. In contrast, alpha3 protein was prominent only when neuronal cells have reached full differentiation. In this model, neuron-depleted cultures did not express the alpha3 isoform, indicating its specificity for mature neuronal cells. UES possessed Na,K-ATPase activity consistent with a single isoform (alpha1), whereas in fully mature neuronal cells a ouabain-sensitive isoform (alpha3) accounted for 27+/-4% of the activity, and a ouabain-resistant isoform (alpha1) 66+/-3%. Immunocytochemistry of mature neuronal cells for alpha1 and alpha3 proteins showed a similar distribution, including cell soma and processes, without evidence of polarization. beta1 protein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cells. While proteins of the beta2 and beta3 isoforms were not detected by immunoblots (except for beta2 in UES), their mRNAs were detected in UES and EB (beta2 and beta3), and in immature and fully differentiated neuronal cells (beta3). Message for the beta2 isoform, however, was not present in neuronal cells. gamma subunit mRNA and protein were undetectable at any stage. These results provide further characterization of neuron-like cells obtained by induction of ES cells in vitro, and establish a model for the expression of isoforms of the Na,K-ATPase during neuronal differentiation. The relation to other aspects of neuronal cell development and relevance to a specialised function for the alpha3 subunit in neurons are discussed.