An abnormal protease-resistant isoform of the protein PrP accumulates in the brain of hosts with transmissible spongiform encephalopathies (TSEs) and appears to be centrally involved in TSE pathogenesis. Studies with scrapie-infected tissue culture cells have indicated that this abnormal PrP is formed from an apparently normal precursor on the plasma membrane or along an endocytic pathway to the lysosomes. Inhibitors of protease-resistant PrP accumulation might serve as tools for studying the basic mechanism of protease-resistant PrP formation and as potential drugs for TSE therapy. Using scrapie-infected neuroblastoma cells to screen for such compounds in vitro, we found that the amyloid binding dye Congo red and certain sulfated glycans potently inhibited the accumulation of protease-resistant PrP in scrapie-infected cells without apparent effects on the metabolism of the normal isoform. The relative potencies of the sulfated glycans corresponded with their previously determined anti-scrapie activities in vivo, suggesting that the prophylactic effects of sulfated polyanions may be due to inhibition of protease-resistant PrP accumulation. Since protease-resistant PrP amyloid is known to contain sulfated glycosaminoglycans, as do other naturally derived amyloids, we hypothesize that these sulfated inhibitors competitively block binding between PrP and endogenous glycosaminoglycans that is important for its accumulation in a protease-resistant, potentially amyloidogenic state. Drugs which interfere with this (pre)amyloid-glycosaminoglycan interaction may be useful for treating a variety of amyloidoses.