We have cloned and characterized the mouse gene encoding the beta subunit of H+, K(+)-ATPase (EC 3.6.1.36). The entire 10.5-kilobase transcription unit of the H+,K(+)-ATPase beta subunit gene was cloned in three overlapping cosmids encompassing approximately 46 kilobases of genomic DNA. A tight cluster of transcription initiation sites has been localized 24-25 nucleotides upstream of the translation start site and 28-29 nucleotides downstream of a TATA-like sequence. The H+, K(+)-ATPase beta subunit gene is split into seven exons encoding predicted structural domains of the beta subunit protein. 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 mouse H+,K(+)-ATPase beta subunit gene is identical to that of the mouse Na+,K(+)-ATPase beta 2 subunit gene. The conservation of genomic organization, together with the high sequence homology, indicates that the mouse H+,K(+)-ATPase beta and Na+,K(+)-ATPase beta 2 subunit genes originated from a common ancestral gene.