We observed a clustering of cholangiocarcinoma in a part of West Virginia. We analyzed the frequency and type of alterations in the p53 tumor-suppressor gene and the K-ras oncogene to determine whether cholangiocarcinomas from this high-incidence area differ from other cholangiocarcinomas at the molecular level. We studied 12 carcinomas of patients from the high-incidence area (West Virginia group), and 15 carcinomas of patients from nearby states (non-West Virginia group). Over-expression of the p53 gene product, accompanying most mutations in the p53 gene, was determined by immunohistochemistry. p53 sequence analysis of exons 5, 6, 7, and 8 of the p53-immunohistochemical-positive carcinomas was also performed. K-ras codon 12 mutations were detected by the polymerase chain reaction and allele-specific oligonucleotide hybridization. Significantly more cholangiocarcinomas from the West Virginia group were p53-immunohistochemical-positive than from the non-West Virginia group (67% vs. 20%; p < 0.05). p53 mutations did not differ in the 2 groups in respect to site or specific type. No differences were found between the 2 groups regarding K-ras mutations (17% vs. 27%). Although the higher frequency of p53-immunohistochemical positivity in the West Virginia group may reflect a different etiology of these cholangiocarcinomas, explaining the high incidence in this area, results of p53 sequence analysis were not different in the West Virginia group. The high incidence may be explained by difference in carcinogenic dose or a different etiology not reflected in p53 or K-ras alterations.