Mutations in the p53 tumor suppressor gene are the most common genetic alterations found in human cancer. Most mutations are accompanied by stabilization of the protein, which renders the mutations detectable through immunohistochemical techniques. The immunoreactivity of p53, however, might not correlate with the result of p53 DNA sequencing. In order to explain the discrepancy, we studied the p53 expressions, mutations, and changes of the three-dimensional protein structure of mutant p53 in a series of 61 pancreatic adenocarcinoma specimens using immunohistochemistry, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), DNA sequencing, and computerized protein modeling. PCR-SSCP followed by DNA sequencing of the p53 gene showed mutations in 31.2% (19 of 61) of the pancreatic adenocarcinomas. Eight of 19 cases showed p53 immunopositivity. These mutations were located on the surface of the three-dimensional structure or formed unfolded proteins, which were easily recognized by the antibody. Among other mutations in which p53 was immunonegative, five cases with deletions and insertion caused frameshift and formation of severely truncated p53 protein structures unreactive with the antibody used. In three cases with point mutations, the mutant amino acids were located in the core of the tightly packed beta sandwich inaccessible to the antibody. Three silent mutations were immunonegative, corresponding with the absence of amino acid changes. These results strongly suggest that the analysis of a computer-generated p53 three-dimensional model based on DNA sequencing data can assist in evaluating the significance of p53 immunostaining and mutations for clinical applications.