Background: Li-Fraumeni syndrome (LFS) is a familial cancer predisposition syndrome that results from germline alterations in the p53 gene. Unlike other cancer predisposition syndromes, LFS predisposes to a wide range of cancers including premenopausal breast cancer, sarcomas, brain tumors, adrenocortical carcinomas, and others. Over the years, the criteria for LFS have expanded beyond the classical definition, resulting in an increased number of individuals being tested. Additionally, technological advances have facilitated discovery of previously occult alterations. Upon review of samples received by our laboratory for p53 analysis between 1991 and 2004, we identified 67 samples for retesting. Methods: We searched our Cancer Genetics database for banked blood-derived DNA samples that had undergone p53 mutation analysis prior to April, 2004 and were found not to have an alteration. Diagnosis and/or family history were confirmed to meet the classical, Birch, or Chompret criteria for LFS. Seventy-three patient samples were retrieved and of those, 67 were of sufficient quality and quantity for analysis. The samples were submitted to the hospital?s Molecular Genetics Laboratory for analysis. Results: Of the 67 samples submitted for p53 analysis, 4/67 were found to harbor a heterozygous p53 germline deleterious mutation (Cys229Arg, Val157Ile, and Tyr236X (n=2)), 1/67 was found to be wildtype, and the remaining 62/67 were found to harbor a presumed non-disease-causing codon 72 polymorphism (Arg72Arg, Pro72Arg). Although these mutations have previously been identified in somatic tissues (www-p53.iarc.fr), this is the first published report of their occurrence in the germline. Conclusions: Technological advances in genetic testing facilitated the identification of a p53 mutation in four individuals who were previously found to be wildtype. Positive findings are important for affected individuals with respect to risk of development of second malignancies and for other at-risk family members, given an overall lifetime cancer risk of 85% in individuals with LFS. These findings stress the importance of banking DNA in anticipation of future advances in technology.