In a highly ouabain-resistant clone from the Madin-Darby canine kidney cell line (Ki > 4 mM), we have previously identified mutations (C113 to Y or C113 to F) in the first transmembrane helix (H1) of the Na,K-ATPase alpha subunit that increase the Ki of a ouabain-sensitive Na,K pump by 1000-fold. Here we report another mutation (Y317 to C) located in the extracellular segment that joins the third and fourth transmembrane domains (H3-H4 ectodomain) of the alpha subunit that also changes ouabain sensitivity of the Na pump. When this mutation (Y317C) was introduced into the Na,K-ATPase alpha 1 subunit of Xenopus laevis, the ouabain inhibition constant increased by a factor of 5, from 130 (wild type) to 800 nM (mutant). However, the expression of double mutants (C113Y + Y317C) in Xenopus oocytes resulted in highly ouabain-resistant Na,K pumps (Ki approximately 7 mM), reproducing the phenotype of the original Madin-Darby canine kidney cell line. When a more conservative change (Y317F) was introduced into the Na,K-ATPase alpha 1 subunit of X. laevis, the ouabain koff increased and expression of double mutants (C113Y + Y317F) resulted in an intermediate ouabain-resistant Na,K pump (Ki approximately 500 microM). We propose that, in addition to the previously identified H1-H2 ectodomain of Na,K-ATPase alpha subunit, the H3-H4 ectodomain also participates in the structure and/or the function of the ouabain binding site. In this respect, the Y317 plays a critical role since the most conservative change Y313 to F is sufficient to significantly affect ouabain binding.