The conformational change of a host protein, PrP^C, into a disease-associated isoform, PrP^Sc, appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt–Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrP^C and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrP^Sc molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrP^Sc molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrP^Sc molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrP^C molecules, copurified lipid molecules, and a synthetic polyanion.