Phospholipase A2 was purified from the pyloric cecum of Asterina pectinifera by delipidation with chloroform-methanol (2:1, v/v) followed by gel filtration on Sephacryl S-200, DEAE-cellulose column chromatography, and gel filtration on Sephadex G-50. The final enzyme preparation was nearly homogeneous in SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 20,000. The enzyme mainly released oleic acid from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. For hydrolysis of egg yolk phosphatidylcholine, the optimum pH and temperature of this enzyme were at around pH 9.0 and 50°C, respectively, and the enzyme was activated by Ca2+. The enzyme rapidly hydrolyzed phosphatidylcholine as the substrate compared with phosphatidylethanolamine. The enzyme did not possess the fatty acid specificity for hydrolysis of phosphatidylcholine prepared from squid mantle muscle.