The lysosomal acid sphingomyelinase is a polymeric glycoprotein (subunit Mr between 28000 and 70000) which hydrolyses natural sphingomyelin, coloured and fluorescent semj-synthetic analogues (trinitrophenylaminolauryl-sphingomyelin and pyrenedecanoyl-sphingomyelin) and the synthetic analogue 2-N-hexadecanoylamido-nitrophenyl-phosphorylcholine. The suitability of these Substrates and of synthetic fluorescent derivatives of methylumbelliferone is discussed. The effect of lipids, detergents and other effectors on the enzyme activity is also described.