Sanfilippo A syndrome (mucopolysaccharidosis type IIIA, MPS-IIIA) is an autosomal recessive neurodegenerative disorder due to an enzymatic defect of the lysosomal enzyme sulphamidase (EC 3.10.1.1) required for the degradation of heparan sulphate. In this study, molecular defects in the sulphamidase gene of MPS-IIIA patients were investigated in a group of 10 patients of Australian and American origin. The entire coding region of the sulphamidase gene was RT-PCR amplified and one polymorphism (R456H), four novel mutations (S66W, R245H, E447K, 1307 del 9) and one previously described mutation (1284 del 11) were identified by direct PCR sequencing. R245H was present in six patients including one severely affected homozygote. In three of the other patients with R245H, second mutant alleles were identified as S66W, 1284 del 11 and E447K, respectively. S66W was also detected in another patient where the other mutant allele remains undefined. In addition, 1307 del 9 was also detected in a patient with the other mutant allele remaining undefined. Allele specific oligonucleotide hybridisation was used to determine the incidence of these in a population of 26 MPS-IIIA patients (Australian and American) and 60 normal controls (Australian). R245H represented 27% (14/52 alleles) in this total patient population, while the other three changes ranged from 1.9 to 9.6% (1-5 of 52 alleles). The sequence variant, R456H, was shown to be polymorphic as it was present in 55% of normal and 38% of patient alleles. The total combined incidence of these five is 46% of alleles. This is the first study of the molecular defects in MPS-IIIA patients and will greatly assist the development of molecular analysis for MPS-IIIA patients and studies concerned with genotype to phenotype relationships.