Basic isozymes of chymotrypsin-like esteroprotease from mouse submandibular glands were purified 60-80-fold by a rather simple procedure consisting of CM-Sepharose CL6B chromatography and gel filtration on Sephadex G-100. The purified sample contained three major isozymes (A, B, C) and some minor ones. Their isoelectric points were between pH 10 and 11. The molecular weights of the main isozymes were estimated at 28000 by SDS-polyacrylamide gel electrophoresis. The acidic isozyme (A) separated into two polypeptide chains whose molecular weights were 21500 and 6500. Specific activities of these isozymes using Bz-Tyr-OEt as substrate were comparable to that of bovine pancreatic alpha-chymotrypsin, but they hydrolyzed casein 10 times slower than did alpha-chymotrypsin. The hydrolytic activities of these isozymes on Bz-Tyr-OEt were inhibited by diisopropylfluorophosphate, tosyl-L-phenylalanine chloromethyl ketone and chymostatin, but they were 400 times less sensitive to chymostatin than was alpha-chymotrypsin.