The cellular prion protein (PrPC) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrPC is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrPC can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrPC was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the -secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrPC could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl--cyclodextrin promoted the shedding of PrPC via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrPC is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrPC can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.