Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopaties (TSEs), which appear to be composed mainly or exclusively of the misfolded prion protein (PrPSc). Prion replication involves the conversion of the normal prion protein (PrPC) into the misfolded isoform, catalyzed by tiny quantities of PrPSc present in the infectious material. We have recently developed the Protein Misfolding Cyclic Amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrPSc. Indeed, after 7 rounds of PMCA we were able to generate large amounts of PrPSc starting from a 1 x10-12 dilution of scrapie hamster brain, which contains the equivalent to approximately 26 molecules of protein monomers. According to recent data this quantity is similar to the minimum number of molecules present in a single particle of infectious PrPSc, indicating that PMCA may enable detection of as little as 1 oligomeric PrPSc infectious particle. Interestingly, the in vitro generated PrPSc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of brain infectious material. The unprecedented amplification efficiency of PMCA leads to several billion folds increase of sensitivity for PrPSc detection as compared to standard tests used to screen prion infected cattle and at least 4000-times more sensitivity than the animal bioassay. Therefore, PMCA offers a great promise for the development of a highly sensitive, specific and early diagnosis of TSE and to further understand the molecular basis of prion propagation.