Cardiac glycosides such as ouabain and digoxin specifically inhibit the Na,K-ATPase. Three new residues in the carboxyl half of the Na, K-ATPase, Phe-786, Leu-793 (PFLIF786IIANIPL793PLGT797), and Phe-863 (FTYF863VIM) have been identified as ouabain sensitivity determinants using random mutagenesis. Polymerase chain reaction was utilized to randomly mutate the DNA sequence encoding the amino acids between Lys-691 and Lys-945 in the alpha subunit of the Na, K-ATPase. This region contains four transmembrane segments (H5, H6, H7, and H8) and the connecting extracellular and cytoplasmic loops. Diverse substitutions of these three residues resulted in proteins displaying 2.8-48-fold increases in the I50 of different cardiac glycosides for inhibition of the Na,K-ATPase activity. By locating these residues, in conjunction with Thr-797 (Feng, J., and Lingrel, J. B (1994) Biochemistry 33, 4218-4224), a new region of the protein containing the H5-H6 hairpin and the H7 transmembrane segment emerges as a major determinant of ouabain inhibition. Thus, a link between the cardiac glycoside binding site and the cation transport sites of the Na,K-ATPase transpires giving a structural base to the cation antagonism of ouabain binding. Furthermore, this link suggests a possible mechanism for cardiac glycoside inhibition of the Na,K-ATPase, such that ouabain binding to the implicated region blocks the movement of the H5 and H6 transmembrane domains which may be required for energy transduction and cation transport.