Metastatic adenocarcinoma of unknown primary site, eg, to lymph nodes, liver, or lung, may originate from many organs. Microscopic differentiation of adenocarcinomas from the pancreaticobiliary and upper gastrointestinal tracts may be difficult because of shared histologic and immunohistologic features. A high prevalence of cytokeratin (CK)17 expression in pancreaticobiliary adenocarcinoma was reported, and preliminary data indicate infrequent or missing expression in gastric adenocarcinoma. The prevalence of CK17 expression in gastric cardiac and esophageal adenocarcinomas has not been studied. We studied CK17 expression in tissue microarrays of 67 distal gastric, 71 gastric cardiac, and 46 esophageal adenocarcinomas and compared it with expression in 55 pancreatic, 23 extrahepatic bile duct, and 49 colorectal adenocarcinomas. CK17 expression was as follows: pancreatic, 88%; bile duct, 59%; esophageal, 30%; distal gastric, 28%; gastric cardiac, 27%; and colorectal adenocarcinoma, 6%. These differences were statistically significant for all tumor types except in comparisons of esophageal, cardiac, and distal gastric adenocarcinoma. The prevalence of CK17 expression in pancreatic and extrahepatic bile duct adenocarcinomas is substantially higher than in upper gastrointestinal tract and colorectal adenocarcinomas. However, in individual cases of adenocarcinoma of unknown primary site, CK17 results alone are insufficient to differentiate the analyzed tumor entities.