We have analyzed the expression pattern of Na,K-ATPase alpha and beta subunit isoforms within the rodent and primate central nervous system. Membrane fractions prepared from rat cerebral cortical type-1 astrocytes and rat cerebellar granule and hippocampal neurons were characterized by immunoblot analyses using a panel of alpha and beta subunit isoform-specific antisera. Each cell type was found to express the alpha 1 isoform but showed differences in the expression of other subunits. Cortical astrocytes displayed alpha 2 and beta 2 subunits, whereas cerebellar granule neurons showed expression of alpha 3 and beta 1 subunits. All three ct subunit isotypes were detected in hippocampal neurons. A survey of the immunofluorescent staining pattern of the alpha 3 subunit in rat and monkey brain confirmed that expression of this Na,K-ATPase alpha subunit isoform was restricted exclusively to neurons. These results suggest that bath neurons and astrocytes express multiple,yet distinct, Na,K-ATPase isoenzymes. The identification of cell types expressing limited combinations of alpha and beta subunits should provide a framework for understanding the physiological significance of Na,K-ATPase isoenzyme diversity and may provide useful tools for the analysis of cell lineage in the mammalian central nervous system.