Clones carrying cDNA sequences for the alpha-subunit of the Na,K-ATPase from HeLa cells have been isolated. Nucleotide sequence analysis of the cloned cDNA has revealed the primary structure of this polypeptide, which consists of 1,023 amino acids. The alpha-subunit of the human Na,K-ATPase exhibited 87% homology with its Torpedo counterpart and 98% homology with its sheep counterpart. The six putative transmembrane segments M1-M6 showed higher conservation than the total segments. Total genomic Southern hybridization indicated the existence of at most two copies, possibly only one, of the gene encoding the Na,K-ATPase alpha-subunit in the human genome.