Tamoxifen (TAM) is a triphenylethylene antiestrogen used for the treatment, and in clinical trials for the prevention, of breast cancer in women. In rats, TAM is a strong liver carcinogen which induces the formation of liver DNA adducts. The DNA of 24 hepatocarcinomas (HCCs) collected at necropsy from individual female Sprague-Dawley rats that were given 22.6 mg/kg TAM daily for 12 months was studied for the presence of mutations in exons 5-9 of the p53 gene by single-strand conformation polymorphism and DNA sequencing analysis. The sequences of introns 5-8 of the rat p53 gene were determined in order to design primers homologous to regions located in these introns. p53 mutations were found in 50% (12 of 24) of the HCCs. These mutations were all specifically clustered in two sites, codons 231 (exon 6-7) and 294 (exon 8). Nine HCCs contained a transition from adenine to guanine in the second base of codon 231 (CAC to CGC), which resulted in a histidine to arginine amino acid substitution; 4 HCCs contained a nonmiscoding transition from cytosine to thymidine in the third base of codon 294 (TGC to TGT; cysteine to cysteine). One HCC contained both mutations. The present report supports previous observations on the genotoxicity of TAM in rodents and raises concerns about its use as a chemopreventive agent against breast cancer in women.