A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.