The cardiac glycoside ouabain inhibits Na,K‐ATPase by binding to the alpha subunit. In a highly ouabain resistant clone from the MDCK cell line, we have found two alleles of the alpha subunit in which the cysteine, present in the wild‐type first transmembrane segment, is replaced by a tyrosine (Y) or a phenylalanine (F). We have studied the kinetics of ouabain inhibition by measuring the current generated by the Na,K‐pump in Xenopus oocytes injected with wild‐type and mutated alpha 1 and wild‐type beta 1 subunit cRNAs. When these mutations, alpha 1C113Y and alpha 1C113F [according to the published sequence [Verrey et al. (1989) Am. J. Physiol., 256, F1034] were introduced in the alpha 1 subunit of the Na,K‐ATPase from Xenopus laevis, the inhibition constant (Ki) of ouabain increased greater than 1000‐fold compared with wild‐type. A more conservative mutation, serine alpha 1C113S did not change the Ki. We observed that the decreased affinity for ouabain was mainly due to a faster dissociation, but probably also to a slower association. Thus we propose that an amino acid residue of the first transmembrane segment located deep in the plasma membrane participates in the structure and the function of the ouabain binding site.