Monoclonal antibody mAbA33 recognizes a unique cell surface protein of colorectal cancer, the A33 antigen. Phase I/II studies have shown highly selective targeting of radio-labeled mAbA33 in patients with metastatic colorectal cancer, opening up the possibility of A33-directed therapy. To better understand the basis of selective mAbA33 targeting in patients, the present study was designed to define the A33 distribution in normal organs and examine A33 expression in a broad range of tumor types. Immunohistochemical analysis of normal tissues identified the large and small intestinal mucosa as the principal sites of A33 expression. Tests with over 450 tumor samples showed that only tumors of the gastrointestinal tract are consistently A33-positive. For example, A33 is found in 95% of primary and metastatic colorectal cancers (86 of 90 cases tested), with uniform expression throughout the tumors in most cases. A33 was detected in a subset of gastric cancers (14/24 cases), with uniform expression in 10 cases (including 5 signer ring adenocarcinomas). Among esophageal cancers, squamous cell tumors typed A33-negative (9 cases), whereas one case each of mucinous and intestinal type was A33-positive. A subset of pancreatic carcinomas (6/12 cases) showed A33 expression, although with marked heterogeneity. Other epithelial cancers, sarcomas, neuroectodermal tumors, and lymphoid neoplasms were generally A33-negative. A33 is the first example of a constitutively expressed, organ-specific epithelial membrane antigen permitting highly specific tumor targeting in patients with colorectal cancer.