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UDP-N-acetylglucosamine (UDP-GlcNAc) is the donor of N-acetylglucosaminyl-1-phosphate (GlcNAcP) in the reaction catalyzed by GlcNAcP transferase, the enzyme deficient in patients with I-cell disease (ICD) and pseudo-Hurler polydystrophy (PHPD). The use of commercially available UDP-[3H or 14C]GlcNAc rather than the synthetically made [β-32P]UDP-GlcNAc was inadequate because of high background. We have overcome this in the assay of GlcNAcP trasferase with α-methylmannoside acceptor by removal of free [3H or 14C]GlcNAc which appeared to be the major breakdown product. In addition, the α-methylmannose-6-phospho-1-[3H or 14C]GlcNAc product of the transfer reaction was then isolated and following desalting could be used as a substrate for the assay of αGlcNAc phosphodiesterase. Using these relatively simple methods, deficiency of GlcNAcP transferase activity could be demonstrated in fibroblasts from patients with the classical forms of ICD (n=4; less than 4% of control activity) and PHPD (n=4; 3-33% of control activity). αGlcNAc phosphodiesterase activity was within the normal range. In contrast, in three related adult patients with what appears to be a very mild form of PHPD, both activities were normal and utilization of natural lysosomal enzyme acceptor (β-glucuronidase) was required to reveal deficiency of GlcNAcP transferase activity.