4‐Methylumbelliferyl‐α‐d‐N‐sulphoglucosaminide (MU‐α‐GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulphamidase. Sanfilippo A patients' fibroblasts (n=42) and lymphocytes (n=1) showed 0–3% of mean normal heparin sulphamidase activity; in total leukocytes from patients (n=8) sulphamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulphamidase activity was reduced in 9 out of 10 cases. Heparin sulphamidase desulphates MU‐αGlcNS to MU‐αGlcNH₂ and further hydrolysis during a second incubation is required to liberate 4‐methylumbelliferone, which can be measured. Yeastα‐glucosidase, which has low but sufficientα‐glucosaminidase activity, was used to hydrolyse the reaction intermediate MU‐αGlcNH₂ to release 4‐methylumbelliferone and free glucosamine.