We have cloned and characterized the mouse Na,K-ATPase beta 2 subunit gene (Atp1b2). The gene spans approximately 7 kb and is split into seven exons. The transcription initiation site has been mapped and consensus TATA and putative CAAT sequences have been found at positions -23 and -137, respectively. Discrete structural domains of the beta 2 subunit protein are encoded by separate exons: The intracellular amino-terminal and putative transmembrane domains are encoded by individual exons and the extracellular carboxyl-terminal domain is encoded by five exons. The exon/intron organization of the beta 2 subunit gene closely resembles that of the H,K-ATPase beta subunit gene, suggesting that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the mouse and rat beta 2 subunit genes reveals a remarkably high degree of homology within a 788-nucleotide segment immediately upstream of the transcription start site. This observation suggests that elements that serve to regulate the cell-specific expression of the beta 2 subunit gene are likely to be located within this conserved region.