Chymotrypsin was capable of hydrolyzing the chromogenic substrate, L-leucyl [beta] naphthylamide. This hydrolysis could be inhibited by the addition of 0.5 mM of diisopropylfluorophos-phate. Chymotrypsin differed from leucine aminopeptidase in being unable to hydrolyze L-leucylglycine, L-leucinamide, and DL-alanyl [beta]- naphthylamide. Neither chymotrypsinogen nor trypsin produced any hydrolysis of L-leucyl [beta] naphthylamide, but the mixture of the two caused hydrolysis of this substrate.