A fluorescent assay for Clostridium Perfringens phospholipase C is described using l-palmitoyl-2-[6(pyren-l-yl)hexanoyl]-snglycero-3-phospho-N-(trinitrophenyl)aminoethanol (PPHTE) as the substrate. This method is based on the decrease of the quenching of pyrene monomer fluorescence when phospholipase C hydrolyzes PPHTE into pyrenediglyceride and phospho(trinitrophenyl)aminoethanol. The hydrolysis of egg lecithin/PPHTE (25:1 molar ratio) substrate by C. perfringens phospholipase C was linear with time for at least 2 rain. Optimal conditions for the hydrolysis by phospholipase C were 50 mM Tris---HCl pH 7.0--30 mM CaCIJ63 /~M egg lecithin and 2.5 #M PPHTE. The Krn and Vma x values for the hydrolysis of egg lecithin/PPHTE vesicles were 28 #M and 280 pmol rain -I, respectively. The detection limit of the assay was 40 mU of C. perfringens phospholipase C. When diglyceride was included into egg lecithin/PPHTE vesicles up to 30 mol% the reaction velocity increased 13-fold. Higher molar proportions of diglyceride were inhibitory. When the hydrolysis of mixtures of different naturally occurring phospholipids and PPHTE was studied egg lecithin was found to be the best substrate. When dipalmitoylphospholipids with different polar head groups were used the reaction velocity decreased in the order egg lecithin ~, dipalmitoylphosphatidylserine > dipalmitoylphosphatidic acid > dipalmitoylphosphatidylcholine > dipalmitoylphosphatidylglyeerol.