Restriction of sodium, potassium adenosine triphosphatase (Na⁺,K⁺-ATPase) to either the apical or basal-lateral membrane domain of polarized epithelial cells is fundamental to vectorial ion and solute transport in many tissues and organs. A restricted membrane distribution of Na⁺,K⁺-ATPase in Madin-Darby canine kidney (MDCK) epithelial cells was found experimentally to be generated by preferential retention of active enzyme in the basal-lateral membrane domain and selective inactivation and loss from the apical membrane domain, rather than by vectorial targeting of newly synthesized protein from the Golgi complex to the basal-lateral membrane domain. These results show how different distributions of the same subunits of Na⁺,K⁺-ATPase may be generated in normal polarized epithelia and in disease states.