The sarcolemmal Na⁺-K⁺ pump, pivotal in cardiac myocyte function, is inhibited by angiotensin II (Ang II). Since Ang II activates NADPH oxidase we tested the hypothesis that NADPH oxidase mediates the pump inhibition. Exposure to 100 nmol/L Ang II increased superoxide-sensitive fluorescence of isolated rabbit ventricular myocytes. The increase was abolished by pegylated superoxide dismutase (SOD); by the NADPH oxidase inhibitor apocynin; and by myristolated inhibitory peptide to -protein kinase C (PKC), previously implicated in Ang II-induced Na⁺-K⁺ pump inhibition. A role for PKC was also supported by an Ang II-induced increase in co-immunoprecipitation of PKC with the receptor for the activated kinase, and with the cytosolic p47phox subunit of NADPH oxidase. Ang II decreased electrogenic Na⁺-K⁺ pump current (Ip) in voltage-clamped myocytes. The decrease was abolished by SOD, by the gp91ds inhibitory peptide that blocks assembly and activation of NADPH oxidase and by PKC inhibitory peptide. Since co-localization should facilitate NADPH-oxidase dependent regulation of the Na⁺-K⁺ pump we examined if there is physical association between the pump subunits and NADPH oxidase. The 1 subunit co-immunoprecipitated with caveolin 3 and with membrane-associated p22phox and cytosolic p47phox NADPH oxidase subunits at baseline. Ang II had no effect on 1/caveolin3 or 1/p22phox interaction, but increased 1/p47phox co-immunoprecipitation. We conclude that Ang II inhibits the Na⁺-K⁺ pump via PKC-dependent NADPH oxidase activation.