In mammalian cells, the Sec61 complex and translocating chain-associated membrane protein (TRAM) are necessary and sufficient to direct the biogenesis, in the appropriate topology, of all secretory and membrane proteins examined thus far. We demonstrate here that the proper translocation of the prion protein (PrP), a substrate that can be synthesized in more than one topologic form, requires additional factors. In the absence of these additional factors, PrP is synthesized exclusively in the transmembrane topology (termed the CtmPrP form) associated with the development of neurodegenerative disease. Thus, translocation accessory factors, acting on some but not other substrates, can function as molecular switches to redirect nascent proteins toward divergent topologic fates with different functional consequences.