A versatile continuous fluorometric assay for phospholipases A2, C, and D has been developed utilizing polymerized mixed liposomes made of pyrene-containing phospholipids (5 mol%) uniformly inserted in the polymerized liposomes of 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphoglycerol (BLPG) and its derivatives. 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine was used for phospholipase A2 and N-(1-pyrenesulfonyl)-egg phosphatidyl ethanolamine for phospholipases C and D. Fluorescence emission of pyrene moieties in polymerized mixed liposomes was strongly quenched by BLPG molecules and, thus, the hydrolysis of pyrene-containing phospholipids and the subsequent displacement of pyrene moieties from the liposomes resulted in a large increase in fluorescence intensity. All the phospholipases tested selectively and rapidly hydrolyzed the inserted pyrene-containing phospholipids, which were readily monitored by measuring an increase in fluorescence emission at 380 nm. Assay conditions for individual phospholipases were optimized by altering interfacial properties of polymerized liposomes, such as surface charge, and subsequently by changing the chemical structure of hydrolyzable phospholipids. Phospholipase activities were linearly proportional to enzyme concentrations in the range from 0.1 to 50 ng. Specific activity determined for phospholipases from a wide variety of sources ranged from 0.5 to 100 mumol/min/mg. Polymerized mixed liposomes are exceptionally stable against chemical and physical degradation and the assay requires only a small amount of pyrene-containing phospholipids. In addition, the polymerized matrix of BLPG (and its derivatives), due to its inertness to the phospholipase hydrolysis, allows the direct measurement of the equilibrium dissociation constant for a protein-liposome complex.