The weight of accumulated evidence suggests a role for the p53 tumor suppressor gene in the development of human hepatocellular carcinoma (HCC). Most striking is an apparent mutational specificity at codon 249 of the human gene. Aflatoxin B1 (AFB) is a liver-specific carcinogen which causes G to T substitutions. This transversion was detected at codon 249 in about 50% of the analyzed HCC tumors from African and Asian patients. In these geographic regions aflatoxin exposure and hepatitis B viral infection are risk factors for HCC. In contrast to the human data, no mutations at codon 249 were detected in AFB-induced tumors from non-human primates. We have analyzed the p53 gene at the site corresponding to codon 249 of the human gene in AFB-induced preneoplastic hepatic nodules from rats. No mutations were detected in the tissues examined. Our data suggest that, at least in the rat, AFB exposure alone may not be sufficient for the specificity of p53 mutations observed in HCC.