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AIM: To characterize the tumor suppressor gene p53 mutations and study the correlation of p53 gene mutation and the expression of P53 protein in cholangiocarcinoma. METHODS: A total of 36 unselected, frozen samples of cholangiocarcinoma were collected. p53 gene status(exon 5-8) and P53 protein were examined by automated sequencing and immunohistochemical staining, combined with the clinical parameters of patients. RESULTS: p53 gene mutations were found in 22 of 36 (61.1%) patients. Nineteen of 36 (52.8%) patients were positive for P53 protein expression. There were significant differences in extent of differentiation and invasion between the positive and negative expression of P53 protein. However, there were no significant differences in pathologic parameters between the mutations and non-mutations. CONCLUSION: The alterations of the p53 gene evaluated by DNA sequence analysis is relatively accurate. Expression of P53 protein could not act as an independent index to estimate the prognosis of cholangiocarcinoma.