The activity of phospholipase A2 (PLA2) on phospholipid liposomes depends on the physicochemical properties of the aggregated substrate, which are subject to continuous modification by the products released during hydrolysis. We propose here an experimental design that, by means of the incorporation of a fluorescent substrate at very low molar ratio (< or = 1:500) into a nonhydrolizable liposomal matrix of 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC), allows the study of hydrolysis by porcine pancreatic phospholipase A2, in virtual absence of physical perturbations of the lamellar phase, by the released products. We have been able to measure immediate hydrolysis of the fluorescent substrate 1,2-di-[omega(1'-pyreno)-decanoyl]-sn- glycero-3-phosphocholine when the sonicated liposomal matrix is in the gel phase. In the liquid crystalline state, in contrast, hydrolysis is very poor even after 80 min of adding the enzyme. Both in the gel and liquid-crystalline phases, incorporation of unlabeled PLA2 products activates the hydrolysis rate to comparable levels. It appears that the conformation adopted by the substrate immersed in the gel or liquid crystalline matrix is especially important in determining its susceptibility to hydrolysis in the absence of products.