Previous studies showed the presence of a significant fraction of Na(+)-K(+)-ATPase alpha-subunits in cardiac myocyte caveolae, suggesting the caveolar interactions of Na(+)-K(+)-ATPase with its signaling partners. Because both alpha- and beta-subunits are required for ATPase activity, to clarify the status of the pumping function of caveolar Na(+)-K(+)-ATPase, we have examined the relative distribution of two major subunit isoforms (alpha(1) and beta(1)) in caveolar and noncaveolar membranes of adult rat cardiac myocytes. When cell lysates treated with high salt (Na(2)CO(3) or KCl) concentrations were fractionated by a standard density gradient procedure, the resulting light caveolar membranes contained 30-40% of alpha(1)-subunits and 80-90% of beta(1)-subunits. Use of Na(2)CO(3) was shown to inactivate Na(+)-K(+)-ATPase; however, caveolar membranes obtained by the KCl procedure were not denatured and contained approximately 75% of total myocyte Na(+)-K(+)-ATPase activity. Sealed isolated caveolae exhibited active Na(+) transport. Confocal microscopy supported the presence of alpha,beta-subunits in caveolae, and immunoprecipitation showed the association of the subunits with caveolin oligomers. The findings indicate that cardiac caveolar inpocketings are the primary portals for active Na(+)-K(+) fluxes, and the sites where the pumping and signaling functions of Na(+)-K(+)-ATPase are integrated. Preferential concentration of beta(1)-subunit in caveolae was cell specific; it was also noted in neonatal cardiac myocytes but not in fibroblasts and A7r5 cells. Uneven distributions of alpha(1) and beta(1) in early and late endosomes of myocytes suggested different internalization routes of two subunits as a source of selective localization of active Na(+)-K(+)-ATPase in cardiac caveolae.