Sanfilippo syndrome type D is an autosomal recessive lysosomal storage disease that is caused by a deficiency of N-acetylglucosamine-6-sulphatase, one of the enzymes involved in the catabolism of heparan sulphate. Only 15 patients have been described in the literature and just two mutations have been reported to date. We present the clinical, biochemical and molecular analysis of two Italian Sanfilippo D families. Novel homozygous mutations were identified in the affected patients from each family: a large intragenic deletion of 8723 bp encompassing exons 2 and 3 in family 1 and a nonsense mutation, Q272X, in family 2. The deletion is the first large intragenic deletion to be reported in any of the four Sanfilippo subtypes, including Sanfilippo type C in which the gene has recently been identified.