Prions are thought to replicate in an autocatalytic process that converts cellular prion protein (PrP^C) to the disease-associated misfolded PrP isoform (PrP^Sc). Our study scrutinizes this hypothesis by in vitro protein misfolding cyclic amplification (PMCA). In serial transmission PMCA experiments, PrP^Sc was inoculated into healthy hamster brain homogenate containing PrP^C. Misfolded PrP was amplified by rounds of sonication and incubation and reinoculated into fresh brain homogenate every 10 PMCA rounds. The amplification depended on PrP^C substrate and could be inhibited by recombinant hamster PrP. In serial dilution experiments, newly formed misfolded and proteinase K-resistant PrP (PrPres) catalyzed the structural conversion of PrP^C as efficiently as PrP^Sc from brain of scrapie (263K)-infected hamsters, yielding an ≈300-fold total amplification of PrPres after 100 rounds, which confirms an autocatalytic PrP-misfolding cascade as postulated by the prion hypothesis. PrPres formation was not paralleled by replication of biological infectivity, which appears to require factors additional to PrP-misfolding autocatalysis.