During the course of the disease, cellular prion protein undergoes conformational conversion into its pathological aggregated form, PrPSc. In humans, prion diseases can be sporadic, acquired or genetic, linked to mutations in the gene encoding prion protein, PRNP. In the BU 4061T globular domain over 20 different mutations in PRNP have been associated with familial forms of prion disease, familial Creutzfeldt-Jakob disease, Gerstmann-Stra¨ussler-Scheinker syndrome, and fatal familial insomnia. Some of these mutations are in the hydrophobic core and about one third of the mutations are substitutions for an amino acid with increased hydrophobicity. Several mechanisms have been proposed to explain how certain point mutations might modulate protein misfolding, such as decreased thermodynamic stability of PrPC, increased stability of the folding intermediate or differences in posttranslational modifications and cellular trafficking such as atypical glycosylation of V180I and T183A. The mechanism of PrPC to PrPSc structural conversion is to a large extent still unknown; particularly since the high resolution structure of PrPSc could not be resolved. Nevertheless, based on biochemical and biophysical characterization of PrPSc aggregates and in vitro prepared PrP fibrils, several structural models of PrPSc were proposed. We recently demonstrated, using disulfide tethering, that the hydrophobic core of the structured Cterminal domain is affected since the subdomains B1-H1-B2 and H2-H3 must separate during PrP conversion. However, disulfide tethers within subdomains did not prevent conversion, suggesting domain swapping as the process underlying PrP conversion. The aim of the present study was to investigate the effect of the enhancement of the hydrophobic core of the H2-H3 globular subdomain on the PrP fibrillization capacity and its conversion into proteinase K resistant PrP. Appropriately selected hydrophobic mutants could increase the stability of the globular domain or its subdomains and we wanted to investigate if increasing the hydrophobicity had any effect on the fibrillization propensities of these types of PrP mutants. Furthermore, cell culture experiment was performed to reveal if PrP mutants could form PrPres and support infection. Three hydrophobic mPrP mutants were selected, prepared and correctly refolded. One of these mutants, T187I, demonstrated increased formation of thin PrP fibrils in vitro and in cell culture increased proteinase K-resistant PrP formation upon 22L prion strain infection despite its increased stability. This mutant might prove as a valuable substrate for in vitro seeding assays and scrapie cell assays. The aim of this study was to investigate the role of hydrophobic mutations in the H2–H3 subdomain of the globular domain of PrP. Several hydrophobic mutations in the globular domain are associated with human prion diseases. Aberrant processing of several of those mutations was observed in cell culture experiments. Several mutants also showed decreased thermodynamic stability of PrPC or increased stability of the folding intermediate when studied in vitro.