The infectious agent (or 'prion') of the transmissible spongiform encephalopathies (TSEs) such as scrapie resembles a virus in that it replicates in vivo and has distinct strains, but it was postulated long ago to contain only protein. More recently, PrPSc, a pathogenic, scrapie-associated form of the host-encoded prion protein (PrP), was identified as a possible primary TSE agent protein. PrPSc is defined biochemically by its insolubility and resistance to proteases and is derived post-translationally from normal, protease-sensitive PrP (PrPc). The conversion seems to involve conformational change rather than covalent modification. However, the conversion mechanism and the relationship of PrPSc formation to TSE agent replication remain unclear. Here we report the conversion of PrPc to protease-resistant forms similar to PrPSc in a cell-free system composed of substantially purified constituents. This conversion was selective and required the presence of preexisting PrPSc, providing direct evidence that PrPSc derives from specific PrPc-PrPSc interactions.