The physiological significance of the cardiac glycoside-binding site on the Na,K-ATPase remains incompletely understood. This study used a gene-targeted mouse (alpha2(R/R)) which expresses a ouabain-insensitive alpha2 isoform of the Na,K-ATPase to investigate whether the cardiac glycoside-binding site plays any physiological role in active Na(+)/K(+) transport in skeletal muscles or in exercise performance. Skeletal muscles express the Na,K-ATPase alpha2 isoform at high abundance and regulate its transport over a wide dynamic range under control of muscle activity. Na,K-ATPase active transport in the isolated extensor digitorum longus (EDL) muscle of alpha2(R/R) mice was lower at rest and significantly enhanced after muscle contraction, compared with WT. During the first 60 s after a 30-s contraction, the EDL of alpha2(R/R) mice transported 70.0 nmol/g.min more (86)Rb than WT. Acute sequestration of endogenous ligand(s) in WT mice infused with Digibind to sequester endogenous cardiac glycoside(s) produced similar effects on both resting and contraction-induced (86)Rb transport. Additionally, the alpha2(R/R) mice exhibit an enhanced ability to perform physical exercise, showing a 2.1- to 2.8-fold lower failure rate than WT within minutes of the onset of moderate-intensity treadmill running. Their enhanced exercise performance is consistent with their enhanced contraction-induced Na,K-ATPase transport in the skeletal muscles. These results demonstrate that the Na,K-ATPase alpha2 isozyme in skeletal muscle is regulated dynamically by a mechanism that utilizes the cardiac glycoside-binding site and an endogenous ligand(s) and that its cardiac glycoside-binding site can play a physiological role in the dynamic adaptations to exercise.