The abnormal prion protein (scrapie-associated prion protein, PrPSc) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrPSc is highly resistant to normal sterilization procedures, the decontamination of PrPSc is a significant public health issue. In the present study, a hyper-thermostable protease, Tk-subtilisin, was used to degrade PrPSc. Although PrPSc is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrPSc under extreme conditions. The level of PrPSc in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 degrees C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrPSc at 100 degrees C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrPSc in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrPSc have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrPSc degradation.