The K-ras oncogene is activated in approximately 90% of pancreatic adenocarcinomas, and the DPC4 (MADH4/SMAD4) tumor suppressor gene is inactivated in approximately 55% of pancreatic adenocarcinomas. The contributions of these genetic alterations to the development of adenocarcinoma of the ampulla of Vater have not been fully established. One hundred forty surgically resected ampullary adenocarcinomas (76 with associated adenomas with high-grade dysplasia) were immunohistochemically labeled for the DPC4 gene product, and in 85 cases the results were correlated with the status of the K-ras oncogene from previously reported data. The results were correlated with clinical outcome and with other pathologic predictors of prognosis. Complete loss of Dpc4 labeling was identified in 34% (95% confidence interval [CI]: 26%, 43%) of the invasive carcinomas and in none (upper 95% CI: 6%) of the associated adenomas. Focal loss of Dpc4 was seen in three (4%; 95% CI: 1%, 14%) of the areas of high-grade dysplasia. Complete loss of Dpc4 expression was seen in 28/77 intestinal-type tumors, in 17/46 pancreaticobiliary-type tumors, and in 0/10 colloid carcinomas. Activating point mutations in the K-ras gene were identified in 40% of the invasive cancers. There was no correlation between K-ras gene mutations and Dpc4 expression and no correlation between these variables and survival. The overall 5-year survival rate was 38%. Lymph node metastases were associated with shorter survival (P =.03). Loss of Dpc4 expression occurs in approximately one third of invasive ampullary cancers but is not seen in adenomas; thus, loss of Dpc4 expression occurs late in ampullary carcinogenesis. Although ampullary and pancreatic adenocarcinomas share histologic and molecular features, ampullary carcinomas are less likely to show loss of Dpc4 expression or K-ras gene mutations.