Phospholipase A2 of rat liver mitochondria was purified approximately 1,400-fold by extraction with KCl, and chromatographies on a Sephadex G-75 column and a diacyl-glycerophosphocholine-Sepharose affinity column. The purified enzyme was very labile when incubated either at 37 degrees C or 0 degrees C, and lost its activity within a few hours. Phospholipids or detergents in the solution protected the enzyme against inactivation. The purified phospholipase A2 preferentially hydrolyzed phosphatidylethanolamine, especially if it contained linoleic acid. The enzyme showed low activity for phosphatidylcholine or phosphatidylinositol.