We have used linkage analysis and fluorescence in situ hybridization to determine the chromosomal organization and location of the mouse (Atp4a) and human (ATP4A) genes encoding the H,K-ATPase alpha subunit. Linkage analysis in recombinant inbred (BXD) strains of mice localized Atp4a to mouse Chromosome (Chr) 7. Segregation of restriction fragment length polymorphisms in backcross progeny of Mus musculus x Mus spretus mating confirmed this assignment and indicates that Atp4a and Atp1a3 (gene encoding the murine Na,K-ATPase alpha 3 subunit) are linked and separated by a distance of approximately 2 cM. Analysis of the segregation of simple sequence repeats suggested the gene order centromere-D7Mit21-D7Mit57/Atp1a3-D7Mit72/Atp 4a. A human Chr 19-enriched cosmid library was screened with both H,K-ATPase alpha and Na,K-ATPase alpha 3 subunit cDNA probes to isolate the corresponding human genes (ATP4A and ATP1A3, respectively). Fluorescence in situ hybridization with gene-specific cosmid clones localized ATP4A to the q13.1 region, and proximal to ATP1A3, which maps to the q13.2 region, of Chr 19. These results indicate that ATP4A and ATP1A3 are linked in both the mouse and human genomes.