A rare conformation of the prion protein, PrP^Sc, is found only in mammals with transmissible prion diseases and represents either the infectious agent itself or a major component of it. The mechanism for initiating PrP^Sc formation is unknown. We report that PrP retrogradely transported out of the endoplasmic reticulum produced both amorphous aggregates and a PrP^Sc-like conformation in the cytosol. The distribution between these forms correlated with the rate of appearance in the cytosol. Once conversion to the PrP^Sc-like conformation occurred, it was sustained. Thus, PrP has an inherent capacity to promote its own conformational conversion in mammalian cells. These observations might explain the origin of PrP^Sc.