Sequencing of p53 exons 5-8 was carried out on 51 initial superficial bladder tumors selected on the basis of high grade and/or p53 overexpression (immunohistochemistry without antigen retrieval). Fourteen point mutations in 13 tumors and one 21 bp deletion in another tumor were identified. In addition, a germ-line mutation corresponding to a previously described polymorphism was detected in exon 6, in two tumors. Mostly G-->A transitions (10) were found. Only three occurred at CpG sites, suggesting a major role for exogenous carcinogens in bladder tumorigenesis. Immunostaining for p53 and MDM2, using antigen retrieval, was carried out on the same tumors. A correlation was found between the percentage of p53-positive cells and the presence of p53 mutations (P = 0.005). No correlation was found between overexpression of p53 and MDM2 in this selected cohort of mostly high grade tumors. The presence of p53 mutations was also analyzed as a function of the smoking habits of the patients. A significant association was found between the presence of p53 point mutations and the number of years of smoking (P = 0.043). All patients with tumors carrying missense or nonsense p53 mutations had smoked for >/=30 years and if former smokers, had stopped for </=5 years. However, no correlation was found between the presence of p53 point mutations and the number of cigarettes smoked. The deletion mutation was the only one present in a tumor from a non-smoker. The data suggest that duration of exposure to carcinogens is the most critical factor in p53 mutagenesis in bladder cancer.