BACKGROUND -CDH17, which is expressed in the intestinal epithelium, is a novel oncogene involved in tumor invasion and metastasis. A panel consisting of cytokeratin (CK) 7, CD20, and CDX2 antibodies is typically used to diagnose gastrointestinal adenocarcinomas. However, studies have shown that CDH17 is a highly specific marker for gastrointestinal adenocarcinoma and may be important in clinical diagnosis. OBJECTIVE -To evaluate the sensitivity and specificity of CDH17, CK20, and CDX2 antibodies in neoplastic tissues, with emphasis on colon, stomach, and esophageal gastrointestinal lineage. METHODS -Immunohistochemistry was performed with CDH17, CK20, and CDX2 antibodies on formalin-fixed, paraffin-embedded tissue microarrays from normal (n = 26) and neoplastic (n = 884) tissues. RESULTS -CDH17 immunostaining was positive in 97.3% (145 of 149) of colon adenocarcinomas, whereas CK20 and CDX2 stained positively in 88.6% (132 of 149) and 93.3% (139 of 149), respectively. In metastatic colon cancers, CDH17, CK20, and CDX2 positive staining was observed in 90.6% (29 of 32), 59.4% (19 of 32), and 81.3% (26 of 32) of cases, respectively. In stomach adenocarcinomas, CDH17 positively stained 64.0% (112 of 175) of tissues, compared to CK20 and CDX2, where staining was observed in only 24.6% (43 of 175) and 46.9% (82 of 175), respectively. In esophageal adenocarcinomas, CDH17, CK20, and CDX2 stained 38.7% (12 of 31), 25.8% (8 of 31), and 29% (9 of 31) of specimens, respectively. Low or no expression was observed in other neoplastic tissues, except pancreatic cancers, where CDH17 displayed higher expression than CK20 and CDX2. CONCLUSIONS -CDH17 is a specific and more sensitive marker in the gastrointestinal tract than CK20 and CDX2. CDH17 may be especially valuable when gastrointestinal tumors are suspected in cancers of unknown primary.