1,2-Bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine was synthesized as a fluorogenic substrate for phospholipase A2. It has a critical micellar concentration of 7.3 μm and gives only excimer fluorescent emission at 480 nm in aqueous micellar dispersion. When hydrolyzed by phospholipase A2, the products give only monomer emission which is monitored best at 382 and 400 nm. Conditions were developed for an assay for phospholipase A2 using this substrate. The assay was sensitive to as little as 8 ng of pure porcine pancreatic phospholipase A2.