During the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two ~10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium- independent phospholipases A2. Detailed inspection of the identified sequence (residues 79,671-109,912 GenBank(TM) accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A2 through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5'-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10-fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0.3 nmol/mg · min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)- 6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC50 = 3 μM), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, and sn-2-lipase activity of a novel intracellular calcium- independent membrane-associated phospholipase A2.