By column chromatography with CM-cellulose, DEAE-Sephadex and Amberlite CG-50 resin, eleven proteolytic enzymes were fractionated from Pronase. They were four neutral proteinases, three alkaline proteinases, three aminopeptidases and carboxypeptidase. Chromatography of Pronase solution on CM-cellulose resolved enzymes into four broad peaks, peaks I, II, III and IV. Peaks I and II were mainly composed of neutral proteinases and aminopeptidases, peak III contained alkaline proteinases and carboxypeptidase and the last peak was alkaline proteinase. Among these enzymes, only the alkaline proteinase present in peak III possessed high activity towards N-benzoyl-L-arginine ethyl ester, N-α-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine-p-nitroanilide. Further separation of aminopeptidase from neutral proteinase contaminated was accomplished by DEAE-Sephadex chromatography. On the other hand, carboxypeptidase was isolated by chromatography on Amberlite CG-50 resin from peak III. The neutral proteinases were inhibited by EDTA, whereas alkaline proteinases were inhibited by DEP but not by EDTA.