In this study, we have followed the fate of newly synthesized cc-and B-subunits of Na,K-ATPase in Xenopus oocytes injected with a and/or B cRNA to examine whether assembly of the two subunits is needed for a correct folding and/or for intracellular transport of Na,K-ATPase. Our data indicate that (1) assembly of a-and /?-subunits occurs at the level of the ER, (2) p-subunits are needed for the newly synthesized a-subunit to adopt a stable configuration and (3) a-and B-subunits mutually depend on each other to be transported out of the ER.