Infection with hepadnaviruses and exposure to dietary aflatoxin are considered major risk factors in the development of hepatocellular carcinoma (HCC) both in humans and in animals. Recently, a broad range of mutations in the p53 tumor suppressor gene has been reported in human HCCs, predominantly from hepatitis B virus carriers in areas with either high or low levels of exposure to dietary aflatoxin. To determine whether p53 mutations are common to HCCs of hosts infected with related hepadnaviruses with and without treatment with aflatoxin, we studied the occurrence of mutations in the p53 gene in HCCs of ground squirrels and woodchucks with history of infection with ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus, respectively. Sequencing of wild type p53 genes from ground squirrels and woodchucks revealed remarkable homology between the two species with only a few amino acid differences in exons 4, 8, and 9. Using direct polymerase chain reaction sequencing, we analyzed the state of the p53 gene (exons 4-9) in 20 HCCs from ground squirrels (2 uninfected, 7 with past, and 11 with ongoing infection with GSHV) and in 11 HCCs from woodchucks persistently infected with woodchuck hepatitis virus. Five GSHV carrier and two uninfected ground squirrels received i.p. administration of aflatoxin B1. We detected only one mutation in the p53 gene of the tested animals. This mutation was located in codon 176 of exon 5 in the HCC of a GSHV-positive ground squirrel treated with aflatoxin. Mutation was caused by a G to T transversion in the second position of the codon, resulting in the replacement of cysteine with phenylalanine, and was accompanied by a tumor-specific loss of heterozygosity. p53 allelic amino acid variation with sequences coding for aspartic acid or asparagine was present in codon 61 in the variable region of exon 4 in both HCCs and nonneoplastic tissues of ground squirrels. In view of the considerably lower apparent rate of mutations in comparison to human HCCs, we suggest a less important role for aflatoxin in the induction of p53 mutations in HCCs of ground squirrels. Alternatively, etiological factors other than p53 mutations may be of greater significance in the development of HCC in ground squirrels and woodchucks.