Novel protease inhibitors were isolated from liquid-cultured mycelia of the white rot fungus Trametes versicolor. Two bands of antiproteinase K activity, TvPI-A and TvPI-B, were detected in the crude cell extract by native polyacrylamide gel electrophoresis (PAGE). Proteins corresponding to TvPI-A were purified by heat treatment, anion-exchange chromatography, and gel filtration. Sodium dodecyl sulfate (SDS)-PAGE demonstrated the presence of three proteins with molecular masses of 14.5, 16.6, and 20 kDa, respectively. T. versicolor protease inhibitors suppressed the activity of proteinase K and, to a smaller extent, of Carlsberg subtilisin, whereas trypsin and chymotrypsins were not inhibited. The inhibitors were acidic proteins and showed remarkable heat stability. To our knowledge, this is the first report about proteinase K inhibitors from fungi.