The infectious agents of prion diseases are composed primarily of an infectious protein designated PrPSc. In cells infected with prions, a host glycoprotein termed PrPC undergoes induced conformational change to PrPSc, but the molecular mechanism underlying this structural transition occurs remains unknown. The prion-seeded conversion of PrPC to protease-resistant PrPSc-like molecules (PrPres) has been studied both in crude and purified in vitro systems in order to investigate the mechanism of protein conformational change in prion disease. Conversion of purified PrPC into PrPres is specific with respect to species-dependent and polymorphic differences in PrP sequence as well as biophysical variations between prion strains, recapitulating the specificity of prion propagation in vitro. The protein misfolding cyclic amplification (PMCA) technique, which utilizes crude brain homogenates, produces much higher yields of PrPres than conversion of purified PrP molecules, suggesting that additional cellular factors may stimulate PrPres formation. In a modified version of the PMCA technique, PrPres from diluted prion-infected brain homogenate can be amplified > ten-fold when mixed with normal brain homogenate without sonication or the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, PrPres amplification in vitro depends upon both time and temperature, has a neutral pH optimum, and does not require divalent cations. In vitro PrPres amplification is inhibited by both reversible and irreversible thiol blockers, indicating that the conformational change from PrPC to PrPres requires a thiol-containing factor. Stoichiometric transformation of PrPC to PrPres in vitro also requires specific RNA molecules, suggesting that host-encoded catalytic RNA molecules may play a role in the pathogenesis of prion disease. Heparan sulfate stimulates conversion of purified PrPC into PrPres in vitro, and heparan sulfate proteoglycan molecules are required for efficient PrPres formation in prion-infected cells. Future studies using in vitro PrPres conversion and amplification assays promise to provide new mechanistic insights about the PrP conversion process, and to generate clinically useful tools.