Werner syndrome (WS) is a rare autosomal-recessive disorder characterized by the premature appearance of features of normal aging in young adults. The extensive phenotypic overlap between WS and normal aging suggests they may also share pathogenetic mechanisms. We reported previously that somatic cells from WS patients demonstrate a propensity to develop chromosomal aberrations, including translocations, inversions, and deletions, and that WS cell lines demonstrate a high spontaneous mutation rate to 6-thioguanine resistance. We report here the biochemical and molecular characterization of spontaneous mutations at the X chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 6-thioguanine-resistant WS and control cells. Blot hybridization analysis of 89 independent spontaneous HPRT mutations in WS and control mutants lacking HPRT activity revealed an unusually high proportion of HPRT deletions in WS as compared with control cells (76% vs. 39%). Approximately half (58%) of the deletions in WS cells consisted of the loss of greater than 20 kilobases of DNA from the HPRT gene. These results suggest that an elevated somatic mutation rate, and particularly deletions, may play pathogenetically important roles in WS and in several associated age-dependent human disease processes.