Gallbladder adenocarcinomas from patients in two high-prevalence areas, Niigata (Japan) and Santiago (Chile), were analyzed for acquired mutations in exons 5-8 of the p53 tumor suppressor gene, and the characteristics of p53 alterations in the two groups were compared. Of 42 tumors, 22 (52.4%) harbored 25 alterations identified by PCR amplification and direct sequencing (13 of 22 tumors from Niigata and 12 of 20 tumors from Santiago). All alterations were single base pair substitutions, 20 (80%) leading to an amino acid substitution or a chain-termination signal, and 5 (20%) were silent. Immunohistochemically, 55 of 84 cases (65.5%) showed overexpression of p53 protein, with no significant difference in frequency between the two areas. Missense mutations correlated highly with overexpression of the p53 protein (93.4%). Mutations of p53 occurred in all four exons examined, most commonly in exon 5, but in no particular "hot spot." In base-change spectra, all 12 mutations from Santiago showed transitions, with 4 arising at the CpG dinucleotide (33.3%). In contrast, no such transition was found at CpG sites in Niigata, and 4 of 13 mutations (30.8%) were transversions. The data indicated that p53 mutations are highly important in carcinogenesis in the gallbladder. In addition, the difference in p53 mutational spectra in Niigata and Santiago indicate a likely regional difference in mutagenesis.