Angiotensin II (Ang II) inhibits the cardiac sarcolemmal Na + -K + pump via protein kinase (PK)C-dependent activation of NADPH oxidase. We examined whether this is mediated by oxidative modification of the pump subunits. We detected glutathionylation of β 1 , but not α 1 , subunits in rabbit ventricular myocytes at baseline. β 1 Subunit glutathionylation was increased by peroxynitrite (ONOO − ), paraquat, or activation of NADPH oxidase by Ang II. Increased glutathionylation was associated with decreased α 1 /β 1 subunit coimmunoprecipitation. Glutathionylation was reversed after addition of superoxide dismutase. Glutaredoxin 1, which catalyzes deglutathionylation, coimmunoprecipitated with β 1 subunit and, when included in patch pipette solutions, abolished paraquat-induced inhibition of myocyte Na + -K + pump current ( I p ). Cysteine (Cys46) of the β 1 subunit was the likely candidate for glutathionylation. We expressed Na + -K + pump α 1 subunits with wild-type or Cys46-mutated β 1 subunits in Xenopus oocytes. ONOO − induced glutathionylation of β 1 subunit and a decrease in Na + -K + pump turnover number. This was eliminated by mutation of Cys46. ONOO − also induced glutathionylation of the Na + -K + ATPase β 1 subunit from pig kidney. This was associated with a ≈2-fold decrease in the rate-limiting E 2 →E 1 conformational change of the pump, as determined by RH421 fluorescence. We propose that kinase-dependent regulation of the Na + -K + pump occurs via glutathionylation of its β 1 subunit at Cys46. These findings have implications for pathophysiological conditions characterized by neurohormonal dysregulation, myocardial oxidative stress and raised myocyte Na + levels.