The isoenzyme of hypoxanthine-guanine phosphoribosyltransferase (HPRT, E.C.2.4.2.8) functions in the metabolic salvage of purines. Partial HPRT deficiency is associated with gouty arthritis, while absence of activity results in Lesch-Nyhan (LN) syndrome. We characterized five unrelated patients with HPRT deficiency to understand the spectrum of molecular defects using Southern and Northern blot, polymerase chain amplification of HPRT mRNA and DNA sequencing, and oligonucleotide hybridization analysis of the HPRT gene. Southern blot analysis of DNA indicated that mutations leading to HPRT deficiency in our five patients were not the result of major chromosomal rearrangements or deletions. Sequencing analysis of the amplified DNA from three different patients with HPRT deficiency implied three unique molecular abnormalities: 1) one single-base substitution at codon 54 (from ATG to CTG) resulting in the replacement of methionine with leucine in an LN patient, 2) two single-base substitutions at codon 179 (from GTT to GGT) and at codon 180 (from GGA to AGA) resulting in the replacement of valine with glycine and glycine with arginine in a gouty patient, and 3) 51 nucleotide deletion between nucleotides 747 and 797 resulting in the formation of shorter sized HPRT mRNA and putative two amino-acid deleted HPRT protein in another gouty patient. These results are the direct molecular evidence of genetic heterogeneity in mutant HPRT.