The γ subunit of the Na,K-ATPase is a small membrane protein that copurifies with the α and β subunits of the enzyme. Strong evidence that the γ subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the γ subunit from several species. The γ subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the γ subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. γ-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl- ouabain (NAB-ouabain), and that this protein, the γ subunit of the Na,K- ATPase, has a distribution pattern along nephron segments that is identical with the α subunit. In addition, coimmunoprecipitation of the α, β and γ subunits demonstrate specific association of the subunits. These results are consistent with the notion that the γ subunit is specifically associated with and may be an important component of the Na,K-ATPase.