We describe the purification and first biochemical characterization of an enzymatic activity in venom from the marine snail Conus magus. This enzyme, named conodipine-M, is a novel phospholipase A2 with a molecular mass of 13.6 kDa and is comprised of two polypeptide chains linked by one or more disulfide bonds. The amino acid sequence of conodipine-M shows little if any homology to other previously sequenced phospholipase A2 enzymes (PLA2s). Conodipine-M thus represents a new group of PLA2s. This is remarkable, since conodipine-M displays a number of properties that are similar to those of previously characterized 14-kDa PLA2s. The enzyme shows little, if any, phospholipase A1, diacyglycerol lipase, triacylglycerol lipase, or lysophospholipase activities. Conodipine-M hydrolyzes the sn-2 ester of various preparations of phospholipid only in the presence of calcium and with specific activities that are comparable to those of well known 14-kDa snake venom and pancreatic PLA2s. The Conus enzyme binds tightly to vesicles of the negatively charged phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphomethanol and catalyzes the hydrolysis of this substrate in a processive fashion. Conodipine-M does not significantly discriminate against phospholipids with unsaturated versus saturated fatty acids at the sn-2 position or with different polar head groups. Linoleoyl amide and a phospholipid analog containing an alkylphosphono group at the sn-2 position are potent inhibitors of conodipine-M. We suggest that the functional resemblance of conodipine-M to other PLA2s might be explained by the utilization of similar catalytic residues.