To investigate the regulation of the Na,K-ATPase, we have studied the expression of the Na,K-ATPase polypeptides in several mammalian cell lines using the vaccinia virus/T7 RNA polymerase expression system. Infection of several fibroblast-like cell lines with viral recombinants containing the Na,K-ATPase α and β isoforms, the glucose transporters, GLUT 1 and GLUT 4, or the capsid protein of the Sindbis virus all result in the production of the appropriate protein products. However, all epithelial cell lines tested fail to synthesize the Na,K-ATPase vital recombinants, yet they efficiently express the other vitally directed polypeptides. While Madin-Darby canine kidney (MDCK) epithelial cells infected with the Na,K-ATPase α1 or β1 recombinant viruses produce both mRNAs, the messages are inefficiently translated. Furthermore, the RNA from infected MDCK cells does not direct the in vitro synthesis of the β1 polypeptide, whereas the message from infected fibroblast-like BSC 40 cells is efficiently translated both in vivo and in vitro. Moreover, the synthesis of the H,K-ATPase γ subunit is also limited in MDCK cells, although the H,K-ATPase β subunit is efficiently expressed. Expression of chimeras constructed between the Na+ pump β1 isoform and the H,K-ATPase β subunit indicates that sequences in the 5' coding region of the β1 message have an inhibitory effect; however, the stringent translational regulation of the β1 isoform in MDCK cells requires the 5' and 3' regions of the coding sequence. The ability of the polarized cell lines to limit the synthesis of the Na+ pump polypeptides while expressing other vaccinia recombinants at high levels suggests that the polarized cells possess a stringent mechanism for the specific translational regulation of a select set of messages.