Conditions for assay of a‐JV‐acetylglucosaminidase activity in human cultured fibroblasts, cultured amniotic fluid cells, leucocytes, serum, plasma and chorionic villi were studied using the fluorogenic substrate 4‐methylumbelliferyl‐2‐acetamido‐2‐deoxy‐a‐D‐glucopyranoside. The substrate was found to have advantage both in terms of sensitivity and ease of use over previously‐used colorimetric substrates for assay of the enzyme in these tissues, and for diagnosis of Sanfilippo B disease and identification of carriers. It should have particular application in first trimester prenatal diagnosis using chorionic villus biopsies.