The conversion of cellular prion protein (PrPC) to the disease-associated misfolded isoform (PrPSc) is an essential process for prion replication. This structural conversion can be modelled in protein misfolding cyclic amplification (PMCA) reactions in which PrPSc is inoculated into healthy hamster brain homogenate, followed by cycles of incubation and sonication. In serial transmission PMCA experiments it has recently been shown that the protease-resistant PrP obtained in vitro (PrPres) is generated by an autocatalytic mechanism. Here, serial transmission PMCA experiments were compared with serial transmission reactions lacking the sonication steps. We achieved approximately 200,000-fold PrPres amplification by PMCA. In contrast, although initial amplification was comparable to PMCA reactions, PrPres levels quickly dropped below detection limit when samples were not subjected to ultrasound. These results indicate that aggregate breakage is essential for efficient autocatalytic amplification of misfolded prion protein and suggest an important role of aggregate breakage in prion propagation. (C) 2004 Elsevier Inc. All rights reserved.