In 6 of 14 neat organic solvents enzymatic activity of dissolved subtilisin was detected. Subtilisin showed approximately 500x higher activity in glycerol than in ethylene glycol, N-methylacetamide, N-methylformamide, 1,2- and 1,3-propanediol. Comparison of soluble proteases in glycerol shows that subtilisin has between 16 to 44x higher V-m, and the same K-m range compared with chymotrypsin, thermolysin, and trypsin using phenyl acetate as a substrate at 30 degrees C. However, the K-m of subtilisin in glycerol was 10x higher than in water. Autolysis rate of subtilisin in glycerol at 30 degrees C is about 3.5x lower than in water, and the rate of autolysis increases from 2.6 to 9x in glycerol and from 44x in water at 37 and 45 degrees C, respectively. Changes in the specificity of bovine serum albumin cleavage by subtilisin were observed by switching from homogeneous glycerol solution to aqueous medium: BSA fragments with different molecular masses in the range of 20 and 40 kDa, and different intensity peaks were detected by using chromatography (fast protein liquid chromatography) and mass spectrometry (matrix-assisted laser desorption ionization-time of flight) techniques.