Peritoneal exudate produced in rat injected with caseinate contained extracellular phospholipase A₂. The activity required Ca²⁺ ion and had a pH optimum of 9 (Chang, H. W., Kudo, I., Hara, S., Karasawa, K., & Inoue, K. (1986) J. Biochem. 100, 1099-1101). This phospholipase A₂ was purified about 14, 000-fold to near homogeneity by the sequential use of column chromatography on Sephadex G-75, Toyopearl HW-65, and TSK ODS-120T reverse-phase HPLC. The final preparation showed a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 13, 500. The enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). When 1-acyl-2-[1-¹⁴C] linoleoyl-sn-glycero-3-phospholipids were used as a substrate, the apparent Km values were 0.027mM with PE, 0.032mM with PS, and 0.1mM with PC, and the Vmax values were 105 μmol/min/mg with PE, 71 μmol/min/mg with PC. The enzyme activity was inhibited by p-bromophenacyl bromide, dithiothreitol, and mepacrine. The amino acid sequence of the NH₂-terminal portion and the amino acid composition of the purified enzyme were deter-mined. They were different from those of rat pancreatic phospholipase A₂, but very similar to those of phospholipase A₂ secreted from rat platelets.