Cardiac glycosides inhibit the Na(+),K(+)-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na(+),K(+)-ATPase are expressed: alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1). It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na(+),K(+)-ATPase isoforms alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1) were expressed in yeast which has no endogenous Na(+),K(+)-ATPase. Isoform specific affinities of digoxin, digitoxin, beta-acetyldigoxin, methyldigoxin and ouabain were assessed in [(3)H]-ouabain binding assays in the absence or presence of K(+) (each n=5). The subcellular localizations of the Na(+),K(+)-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K(+), methyldigoxin (alpha(1)>alpha(3)>alpha(2)) and ouabain (alpha(1)=alpha(3)>alpha(2)) showed distinct isoform specific affinities, while for digoxin, digitoxin and beta-acetyldigoxin no differences were found. In the presence of K(+), also digoxin (alpha(2)=alpha(3)>alpha(1)) and beta-acetyldigoxin (alpha(1)>alpha(3)) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only alpha(1)beta(1) and alpha(2)beta(1) are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na(+),K(+)-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.