We have cloned and characterized the Na,K-ATPase beta 3 subunit gene (ATP1B3), and a beta 3 subunit pseudogene (ATP1B3P1), from a human PAC genomic library. The beta 3 subunit gene is > 50 kb in size and is split into 7 exons. The exon/intron organization of the beta 3 subunit gene is identical to that of the Na,K-ATPase beta 3 subunit gene, indicating that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the human and mouse beta 3 subunit gene reveals a high degree of homology within a 300-bp segment located immediately upstream of the translation start site, suggesting that control elements that serve to regulate the cell-specific expression of the beta 3 subunit gene are likely to be located within this conserved region. Dot blot analysis of beta 3 subunit transcripts revealed expression within virtually all human tissues, while in situ hybridization showed expression of beta 3 mRNA in both neurons and glia of rat brain. Fluorescence in situ hybridization with PAC DNA clones localized ATP1B3 to the q22-->23 region of Chromosome (Chr) 3, and the beta 3 pseudogene to the p13-->15 region of Chr 2.