The cell-surface expression and function of multisubunit plasma membrane proteins are regulated via interactions between catalytic subunits and auxiliary subunits. Subunit assembly in the endoplasmic reticulum is required for the cell-surface expression of the enzyme, but little is known about subunit interactions once it reaches the plasma membrane. Here we performed highly quantitative analyses of the catalytic (α1) and auxiliary (β1 and β3) subunits of Na⁺/K⁺-ATPase in the HeLa cell plasma membrane using isoform-specific antibodies and a cell-surface protein labeling procedure. Our results indicate that although the β-subunit is required for the cell-surface expression of the α-subunit, the plasma membrane contains more α-subunits than β-subunits. Pulse-labeling and chasing of the cell-surface proteins revealed that degradation of the β-subunits was much faster than that of the α1-subunit. Ubiquitylation, as well as endocytosis, was involved in the fast degradation of the β1-subunit. Double knockdown of the β1- and β3-subunits by RNAi resulted in the disappearance of these β-subunits but not the α1-subunit in the plasma membrane. All these results indicate that the α- and β-subunits of Na⁺/K⁺-ATPase are assembled in the endoplasmic reticulum, but are disassembled in the plasma membrane and undergo different degradation processes.