The Na,K-ATPase contains a binding site for cardiac glycosides, such as ouabain, digoxin, and digitoxin, which is highly conserved among species ranging from Drosophila to humans. Although advantage has been taken of this site to treat congestive heart failure with drugs such as digoxin, it is unknown whether this site has a natural function in vivo. Here we show that this site plays an important role in the regulation of blood pressure, and it specifically mediates adrenocorticotropic hormone (ACTH)-induced hypertension in mice. We used genetically engineered mice in which the Na,K-ATPase alpha2 isoform, which is normally sensitive to cardiac glycosides, was made resistant to these compounds. Chronic administration of ACTH caused hypertension in WT mice but not in mice with an ouabain-resistant alpha2 isoform of Na,K-ATPase. This finding demonstrates that the cardiac glycoside binding site of the Na,K-ATPase plays an important role in blood pressure regulation, most likely by responding to a naturally occurring ligand. Because the alpha1 isoform is sensitive to cardiac glycosides in humans, we developed mice in which the naturally occurring ouabain-resistant alpha1 isoform was made ouabain-sensitive. Mice with the ouabain-sensitive "human-like" alpha1 isoform and an ouabain-resistant alpha2 isoform developed ACTH-induced hypertension to greater extent than WT animals. This result indicates that the cardiac glycoside binding site of the alpha1 isoform can also mediate ACTH-induced hypertension. Taken together these results demonstrate that the cardiac glycoside binding site of the alpha isoforms of the Na,K-ATPase have a physiological function and supports the hypothesis for a role of the endogenous cardiac glycosides.