Potassium and sodium displacements across the skeletal muscle membrane during exercise may cause fatigue and are in part controlled by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for muscle functioning. We investigated the effect of oxidative stress (glutathionylation) on Na,K-ATPase activity. Ten male subjects performed three bouts of 4-min submaximal exercise followed by intense exercise to exhaustion with and without beta2-adrenergic stimulation with terbutaline. Muscle biopsies were obtained from m. vastus lateralis at rest (Control samples) and at exhaustion. In vitro glutathionylation reduced (P < 0.05) maximal Na,K-ATPase activity in a dose-dependent manner. Na,K-ATPase α subunits, purified by immunoprecipitation and tested by glutathione (GSH) antibodies, had a basal glutathionylation in Control samples and no further glutathionylation with exercise and beta2-adrenergic stimulation. Immunoprecipitation with an anti-GSH antibody and subsequent immunodetection with β1 antibodies showed approximately 20% glutathionylation in Control samples and further glutathionylation after exercise (to 32%) and beta2-adrenergic stimulation (to 38%, P < 0.05). Combining exercise and beta2-adrenergic stimulation raised the β1 glutathionylation to 45% (P < 0.05). In conclusion, both α and β1 subunits of the Na,K-ATPase were glutathionylated in Control samples, which indicates that the maximal Na,K-ATPase activity is overestimated if based on protein density only. β1 subunits are further glutathionylated by exercise and beta2-adrenergic stimulation. Our data suggest that glutathionylation contributes to the complex regulation of Na,K-ATPase function in human skeletal muscle. Glutathionylation of the Na,K-ATPase may explain reductions in maximal Na,K-ATPase activity after exercise, which may be involved in muscle fatigue.