Glucosamine, galactosamine, mannosamine, several disaccharides and a tetrasaccharide were evaluated as substrates for the N-acetyltransferase involved in the pathogenesis of the Sanfilippo C syndrome. Glucosamine and alpha-D-glucosaminide disaccharides and a tetrasaccharide derived from heparin were exo-N-acetylated by homogenates of cultured skin fibroblast from normal individuals at pH 6.0 in the presence of acetyl-CoA, whereas fibroblast homogenates prepared from a Sanfilippo C patient failed to catalyse the N-acetyltransferase from acetyl-CoA to these substrates. The apparent Km values of the glucosamine and alpha-glucosaminide disaccharide N-acetyltransferase were 98 and 200 mumol/l respectively; the corresponding V values were 200 and 180 nmol.min-1.g-1 fibroblast whole cell homogenate protein respectively. Incubation of homogenates from normal individuals or the Sanfilippo C patient with glucosamine 6-phosphate and acetyl-CoA at pH 6.0 produced N-acetylglucosamine 6-phosphate. Acetyltransfer to glucosamine or glucosamine 6-phosphate in homogenates of normal fibroblasts was not inhibited by the addition of arylamines. It is proposed that N-acetyltransferase to glucosamine, glucosamine 6-phosphate and arylamines is carried out by separate enzymes. Glucosamine is a suitable substrate for the diagnostic assay of the enzyme involved in the exo-N-acetylation of alpha-glucosaminide residues at the non-reducing end of the heparan sulfate stored and excreted by Sanfilippo C patients.