Abstract : We purified an 80‐kDa Ca²⁺‐independent phospholipase A₂ (iPLA₂) from rat brain using octyl‐Sepharose, ATP‐agarose, and calmodulin‐agarose column chromatography steps. This procedure gave a 30,000‐fold purification and yielded 4 μg of a near‐homogeneous iPLA₂ with a specific activity of 4.3 μmol/min/mg. Peptide sequences of the rat brain iPLA₂ display considerable homology to sequences of the iPLA₂ from P388D₁ macrophages, Chinese hamster ovary cells, and human B lymphocytes. Under optimal conditions, the iPLA₂ revealed the following substrate preference toward the fatty acid chain in the sn‐2 position of phosphatidylcholine : linoleoyl > palmitoyl > oleoyl > arachidonoyl. The rat brain iPLA₂ also showed a head group preference for choline ≥ ethanolamine ≫ inositol. The iPLA₂ is inactivated when exposed to pure phospholipid vesicles. The only exception is vesicles composed of phosphatidylcholine and phosphatidylinositol 4,5‐bisphosphate. Studies on the regional distribution and ontogeny of various phospholipase A₂ (PLA₂) types in rat brain indicate that the iPLA₂ is the dominant PLA₂ activity in the cytosolic fraction, whereas the group IIA secreted PLA₂ is the dominant activity in the particulate fraction. The activities of these two enzymes change during postnatal development.