A calcium-independent phospholipase A₂ (iPLA₂) was identified from the cytosolic fraction of rat liver cells. On gel filtration chromatography, the iPLA₂ activity was eluted as broad peaks of 150 to 500 kDa. The enzyme was maximally active at pH 7.5, retained 75% of its original activity after heating at 50℃ for 5 h, and was inhibited by Ca^(2+), Mg^(2+), and Zn^(2+) ions, but was not affected by Na+ and K+ ions. The enzymatic activity was increased up to 150% by 1 to 4 mM DTT and was inhibited up to 25% by 0.1 to 1 mM PMSF. The iPLA₂ activity had preference for the head group of phospholipass, where phosphatidylethanolamine was preferred to phosphatidylcholine. The results suggest that the iPLA₂ may be a novel enzyme distinct from the previously reported iPLA₂s.