Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrP^res) that replicates in the body by inducing the misfolding of the cellular prion protein (PrP^C). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrP^C → PrP^res conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrP^res. The in vitro-generated forms of PrP^res share similar biochemical and structural properties with PrP^res derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrP^res led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.