Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28 000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of Nα-p-Tosyl--arginine methyl ester hydrochloride were approximately pH 8.0 and 55 °C, respectively. A. pectinifera trypsin was unstable at above 50 °C and below pH 5.0, and was not activated by adding Ca2+. The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found.