In this fluorometric assay for phospholipase A2 (EC 3.1.1.4) in serum we use the fluorescent phospholipid analog 1-octacosanyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphatid yl monomethyl ester as substrate. The optimized conditions are: 28 mumol of the substrate per liter of Tris buffer (20 mmol/L, pH 7.4). The hydrolytic reaction is allowed to proceed for 30 min at 37 degrees C. The fluorescent reaction product, (pyren-1-yl)hexanoic acid, is then separated from the unreacted substrate by liquid-liquid phase partition. The concentration of the liberated fatty acid analog is determined fluorometrically. The detection limit is approximately 6 pmol min-1 mL-1. Forty duplicate samples can be assayed in about 2 h. The activity of this enzyme in serum of 20 healthy volunteers averaged 69 pmol min-1 mL-1. For seven patients with clinically diagnosed pancreatitis the average activity was 1092 pmol min-1 mL-1.