We have isolated and characterized cDNA clones encoding the human and rat Na,K-ATPase beta3 subunit isoform. The human cDNA encodes a polypeptide of 279 amino acids that exhibits primary sequence and secondary structure similarities to Na,K-ATPase beta subunit isoforms. Sequence comparisons showed that the human beta3 subunit closely resembles the beta3 subunit of Xenopus laevis (59% amino acid identity) and is less similar to the human Na,K-ATPase beta1 and beta2 subunits (38% and 48% amino acid identity, respectively). By analyzing the segregation of restriction fragment length polymorphisms among recombinant inbred strains of mice, we localized the beta3 subunit gene to murine chromosome 7. Northern blot analysis revealed that the beta3 subunit gene encodes two transcripts that are expressed in a variety of rat tissues including testis, brain, kidney, lung, stomach, small intestine, colon, spleen, and liver. Identification of the mammalian beta3 subunit suggests an even greater potential for Na,K-ATPase isoenzyme diversity than previously realized.