A virtually complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT) causes Lesch-Nyhan syndrome. A novel mutation of HPRT gene in a Japanese Lesch-Nyhan family has been identified using mRNA and genomic DNA from peripheral blood cells. A single nucleotide substitution of T to C in exon 3 resulted in a mis-sense mutation, CTC (Leu) to CCC (Pro), at codon 65. Utilizing an Mn/I restriction site which was lost in the mutation as an indicator, a family study showed that the mother was normal not having the mutant gene. The mutation was a de novo event that had occurred in the germ cells of the mother or in the proband during the early phase of fetal development.