There is currently no effective therapy for human prion diseases. However, several polyanionic glycans, including pentosan sulfate and dextran sulfate, prolong the incubation time of scrapie in rodents, and inhibit the production of the scrapie isoform of the prion protein (PrP_Sc), the major component of infectious prions, in cultured neuroblastoma cells. We report here that pentosan sulfate and related compounds rapidly and dramatically reduce the amount of PrP_C, the non-infectious precursor of PrP_Sc, present on the cell surface. This effect results primarily from the ability of these agents to stimulate endocytosis of PrP_C, thereby causing a redistribution of the protein from the plasma membrane to the cell interior. Pentosan sulfate also causes a change in the ultrastructural localization of PrP_C, such that a portion of the protein molecules are shifted into late endosomes and/or lysosomes. In addition, we demonstrate, using PrP-containing bacterial fusion proteins, that cultured cells express saturable and specific surface binding sites for PrP, many of which are glycosaminoglycan molecules. Our results raise the possibility that sulfated glycans inhibit prion production by altering the cellular localization of PrP_C precursor, and they indicate that endogenous proteoglycans are likely to play an important role in the cellular metabolism of both PrP_C and PrP_Sc.