A proteinase was purified from an extract of the koji culture of a strain of Aspergillus sydowi, isolated from "Katsuobushi", in a homogeneous state by polyacrylamide gel electrophoresis by ammonium sulfate fractionation, and Butyl-Toyopearl, DEAE-Toyopearl and Bio-Gel P-100 column chromatographies. The molecular weight of the enzyme was calculated to be about 40,000. The optimum pH activity was 7.0-7.5 at 37 degrees C and the optimum temperature was 45 degrees C at pH 7.2 for caseinolytic activity. The enzyme was stable between pH 3.5-8.0 and a temperature of up to 45 degrees C. As the enzyme activity was completely inhibited with phenylmethylsulfonylfluoride, this enzyme was a kind of serine proteinase. The enzyme had a different substrate specificity from other fungal serine proteinases reported so far from the results of cleavage of proangiotensin and oxidized insulin B-chain.