A chymotrypsin-like endopeptidase has been purified by ion-exchange chromatography, affinity chromatography, and gel filtration. The enzyme preparation is homogeneous in the ultracentrifuge and disc electrophoresis. The enzyme is proved to be free from any other proteolytic activities. The molecular weight of the proteinase as determined with several techniques (ultracentrifugation, gel filtration and electrophoresis without dodecylsulfate) is in the range between 13000–14000; however, by dodecylsulfate gel electrophoresis a molecular weight of 23000 was obtained. The obtained physical constants of hornet chymotrypsin are: sedimentation coefficient s020,w= 1.96 × 10−13 S, diffusion coefficient D020,w= 131 μm2s−1, partial specific volume v̄= 0.737 ml g−1, frictional ratio f/fmin= 1.04, and degree of hydration = 0.1 g per g protein.