During the process of cultivation of Th. vulgaris several proteases are formed. In the present investigation the extensively purified major component was used. The substrate specificity was determined by means of 7 proteins, 7 amino acid esters, 5 fatty acid esters and 15 amino acid 4-nitroanilides. Among the protein substrates tested, urea denaturated hemoglobin was split best, followed by gelatin, casein, field bean protein, serum albumin and gluten. The weakest rate of hydrolysis was observed with elastin. In contrast to this acetyl-(L-ala)3-methylester, that is a substrate for elastase, was split best from all the esters tested. Only 8% of this activity could be found with the chymotrypsin substrates acetyl-L-tyr-ethylester and acetyl-L-phe-ethylester and 1% of the above activity with the trypsin substrates tosyl-L-arg-methylester and benzoyl-L-arg-methylester. The fatty acid esters and the p-nitroanilides were hydrolyzed much more slowly. The pH-optimum of thermitase was found in the weakly alkaline region of pH 7 to 9. There were only small differences between the individual high and low molecular substrates. The temperature optimum was between 60 and 75 degrees C for esters and p-nitroanilides as substrates and at 90 degrees C for casein. It should be mentioned that the enzyme was quickly inactivated at temperatures above 70 degrees C.