Recent attention has been drawn to the diagnostic potential of tests based on shed colonic tumor markers. Adnab-9 monoclonal antibody raised against neoplastic, potentially premalignant colonic adenomas recognizes a marker in colonic effluent or tissue which correlates with the presence of tumors or risk of colorectal cancer. The origin of this antigen and optimal collection of colonic effluent were investigated by enzyme-linked immunosorbent assay and Western blotting. Mean Adnab-9 binding in effluent samples from colorectal cancer patients even after resection is high as compared with that in normal subjects (P < 0.05). Effluent samples are best collected in the morning hours. Antigen proteolysis may be significant depending on the site and timing of effluent collection, but breakdown products are reactive. Tissue and effluent Adnab-9 binding at any one anatomic site of collection appear to correlate (r = 0.88, P = 0.01). The Adnab-9 antigen is constitutively expressed at low levels throughout the distal bowel and localized to the deepest regions of the mucosal crypts. Other than meconium, no significant levels of binding are found in other body fluids. This antigen is specific for the gastrointestinal tract, its binding in conveniently collected effluent samples correlates with tissue content, and the antigen is constitutively expressed in the crypts of the distal small bowel and colonic mucosa.