The biogenesis of hamster brain prion protein (PrP) has been studied by expression of RNA transcribed from a full-length PrP cDNA in Xenopus oocytes and cell-free systems. Earlier studies in the wheat germ cell-free system showed that one form of PrP is a transmembrane protein that spans the bilayer at least twice [Hay, B., Barry, R. A., Lieberburg, I., Prusiner, S. B., & Lingappa, V. R. (1987) Mol. Cell. Biol. 7, 914-920]. We now report that PrP can also exist as a secreted protein. SP6 PrP RNA microinjected into Xenopus oocytes produced two forms of PrP: one that remained in the cell and another that was secreted into the medium. Cell-free translation studies in rabbit reticulocyte lysates supplemented with microsomal membranes gave similar results: while one form of PrP was found as an integral membrane protein spanning the membrane at least twice, another form of PrP was found to be completely translocated to the microsomal membrane vesicle lumen. Both the membrane and secretory forms of PrP appear to be generated from the same pool of nascent chains. The mechanism governing the alternative fates of nascent PrP remains to be elucidated but may have significance for understanding the pathogenesis of scrapie and other prion diseases.