Fluorescent glycolipids were utilized for detection of the intracellular, activator-dependent, activities of beta-glucocerebrosidase and arylsulphatase A. Activities were measured in primary skin fibroblasts from normal individuals, from patients with Gaucher disease who had mutations within the beta-glucocerebrosidase gene, and from a prosaposin-deficient patient. Fluorescent microscopy demonstrated that glucosylceramide or sulphatide labelled with a fluorescent probe (lissamine-rhodamine) were endocytosed and reached the lysosomes. There, in the presence of active enzyme and the corresponding saposin, they were hydrolysed to fluorescent ceramide, which changed its intracellular localization. When these substrates were labelled with pH-sensitive lissamine-rhodamine, which loses its fluorescence at neutral or alkaline pH, the transport of the product, i.e. fluorescent ceramide, from the lysosomes resulted in disappearance of the cellular fluorescence. In cells of patients having mutations within the genes encoding the glucocerebrosidase or the prosaposin, there was a considerable reduction in the intracellular rate of substrate hydrolysis that could be followed by fluorescence microscopy or measured quantitatively in cell extracts.