Radioactive substrates are being used increasingly in assays for enzymes that are present at very low levels of activity, particularly if artificial chromogenic or fluorogenic substrates are unavailable, or have varying sensitivity towards different isoenzymes. Frequently, these radioactive substrates are commercially unavailable, and must be prepared. Over the past few years fluorogenic substrates (based on 4-methylumbelliferyl derivates) have become available for the assay of the lysosomal enzymes associated with glycosphingolipid catabolism. However, it has recently been shown that such assays may be diagnostically misleading and more consideration is now placed on the use of suitably radiolabelled naturally occurring lipids (corresponding to those accumulated in lysosomal disease states) as substrates in aqueous in vitro assay system. Six of these lipids are of immediate importance—sphingomyelin (accumulated in Niemann-Pick disease), galactosylceramide (Krabbe's), glucosyl-ceramide (Gaucher's), galactosyl-sulphate-ceramide (metachromatic leukodystrophy), GM1 ganglioside (generalized gangliosidosis) and GM2 ganglioside (Tay Sach's). There are 2 principal methods of labelling these lipids to render them suitable for use as substrates. The first involves incorporation of tritium into the water soluble hydrophilic part of the molecule (e.g. terminal carbohydrate or phosphoryl-choline residue). The water soluble label released by enzyme action may then be partitioned from the hydrophobic substrate by relatively simple means although more complex systems must be used when GM1 or GM2 gangliosides are used as substrates. Alternatively, the hydrophobic part of the molecule may be labelled by catalytic tritiation. In our hands, more than 80% of the tritium incorporated goes into the fatty acids of the ceramides, with the remaining 20% to the long chain base. After enzymatic cleavage, the labelled, hydrophobic ceramide may be rapidly separated from the unchanged hydrophobic substrate by chromatography on micro silica gel plates. As before, however, the use of GM1 and GM2 gangliosides require more complicated procedures. Problems which have been overcome in using these tritiated lipids include their limited ‘solubility’ in aqueous assay systems and their non-specific adsorption of silica gel, which necessitates special precautions prior to scintillation counting. Radioactive substrates are being used increasingly in assays for enzymes that are present at very low levels of activity, particularly if artificial chromogenic or fluorogenic substrates are unavailable, or have varying sensitivity towards different isoenzymes. Frequently, these radioactive substrates are commercially unavailable, and must be prepared. Over the past few years fluorogenic substrates (based on 4-methylumbelliferyl derivates) have become available for the assay of the lysosomal enzymes associated with glycosphingolipid catabolism. However, it has recently been shown that such assays may be diagnostically misleading and more consideration is now placed on the use of suitably radiolabelled naturally occurring lipids (corresponding to those accumulated in lysosomal disease states) as substrates in aqueous in vitro assay system. Six of these lipids are of immediate importance—sphingomyelin (accumulated in Niemann-Pick disease), galactosylceramide (Krabbe's), glucosyl-ceramide (Gaucher's), galactosyl-sulphate-ceramide (metachromatic leukodystrophy), GM1 ganglioside (generalized gangliosidosis) and GM2 ganglioside (Tay Sach's). There are 2 principal methods of labelling these lipids to render them suitable for use as substrates. The first involves incorporation of tritium into the water soluble hydrophilic part of the molecule (e.g. terminal carbohydrate or phosphoryl-choline residue). The water soluble label released by enzyme action may then be partitioned from the hydrophobic substrate by relatively simple means although more complex systems must be used when GM1 or GM2 gangliosides are used as substrates. Alternatively, the hydrophobic part of the molecule may be labelled by catalytic tritiation. In our hands, more than 80% of the tritium incorporated goes into the fatty acids of the ceramides, with the remaining 20% to the long chain base. After enzymatic cleavage, the labelled, hydrophobic ceramide may be rapidly separated from the unchanged hydrophobic substrate by chromatography on micro silica gel plates. As before, however, the use of GM1 and GM2 gangliosides require more complicated procedures. Problems which have been overcome in using these tritiated lipids include their limited ‘solubility’ in aqueous assay systems and their non-specific adsorption of silica gel, which necessitates special precautions prior to scintillation counting.