Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP^Sc, the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529–14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 μg of PPI generation 4.0/ml for 4 weeks not only reduced PrP^Sc to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the β-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP^Sc to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP^Sc from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP^Sc from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP^Sc clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.