A monoclonal antibody to colon carcinoma mucin was found to react with a colon carcinoma-associated carbohydrate epitope. This antibody was used to develop a quantitative solid phase immunoassay, M43. We prospectively and retrospectively evaluated the assay in patients with and without gastrointestinal carcinoma and compared the sensitivity and specificity with that of carcinoembryonic antigen (CEA) and CA 19-9. One hundred ninety-two patients (181 with no evidence of malignancy) referred for upper or lower gastrointestinal endoscopy were prospectively studied. Sera from 172 patients with histologically confirmed gastrointestinal adenocarcinoma were retrospectively studied. Optimal discrimination cutoffs for M43 (5 units/ml), CEA (5 ng/ml), and CA 19-9 (30 units/ml) were determined by receiver operating characteristic curves analysis. M43 was positive in 112 of 151 patients with colorectal carcinoma (sensitivity 74%) and was negative in 167 of 181 patients without carcinoma (specificity 92%). Sensitivity and specificity were 77% and 93% for CEA and 60% and 83% for CA 19-9. Sixty-four % of 73 patients with colorectal carcinoma limited to the bowel wall had a positive M43 with a mean value of 178 units/ml. Eighty-one % of 27 patients with nonhepatic metastasis had a positive M43 with a mean value of 223 units/ml. Eighty-four % of 51 patients with hepatic metastasis had a positive M43 assay with a mean value of 2532 units/ml. Sensitivity in these three groups was 67%, 82%, and 82%, respectively, for CEA and 43%, 68%, and 79%, respectively, for CA 19-9. Of 38 carcinoma patients with a negative CEA, 45% had a positive M43. No correlation between the levels of M43 and CEA in patients with colorectal carcinoma was found. We conclude that M43 is positive in most patients with colorectal carcinoma, even in early stages. As a diagnostic test, its sensitivity and specificity are equivalent to those of CEA. However, the M43 assay is measuring a tumor antigen which is fundamentally different from CEA and which is present in a high percentage of CEA-negative patients.