Trypsins from the pyloric ceca of two fish species, yellow tail (Seriola quinqueradiata) and brown hakeling (Physiculus japonicus) were purified by a series of chromatographic separations. Purity increased 62- and 106-fold with approximately 55 and 10% yield for yellow tail trypsin and brown hakeling trypsin, respectively. Final enzyme preparations were homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weights of both enzymes were estimated to be 24 kDa by SDS-PAGE. Yellow tail and brown hakeling trypsins had maximal activity at pH 8.0 for hydrolysis of N?-p-tosyl-L-arginine methyl ester hydrochloride and was unstable at acidic pH. The optimum temperatures for yellow tail and brown hakeling trypsins were 60 and 50C, respectively. Yellow tail trypsin was stable up to 50C, whereas brown hakeling was stable up to 40C. Both trypsins were stabilized by calcium ions. The activities of both trypsins were strongly inhibited by soybean trypsin inhibitor and N -p-tosyl-L-lysine chloromethyl ketone hydrochloride, and were partially inhibited by ethylenediaminetetraacetic acid. The N-terminal amino acid sequences of yellow tail trypsin and brown hakeling trypsin were determined as IVGGYECTPYSQPHQVSLNS and IVGGYECPKHSQPHQVSLNS, respectively.