Monoclonal antibody (MAb) B72.3 was generated against a carcinoma metastasis and has been shown to bind with a high degree of selectivity to a tumor-associated glycoprotein (TAG-72) found in human colon and breast carcinomas, while showing minimal reactivity to any normal adult tissues. Competition radioimmunoassays have been developed for the detection of TAG-72 in tumors and sera from both athymic mice bearing human carcinoma xenografts and patients with colon, breast and other carcinomas. The distribution of TAG-72 in human tumor xenografts was restricted to tumors originating from the LS-174T human colon carcinoma, with no significant reactivity being detected in human tumor xenografts from a different colon carcinoma, a human breast carcinoma, or a human melanoma. The levels of TAG-72 in clinical material obtained from surgery were examined; high levels were found in colon carcinomas and to a lesser extent in breast carcinomas, while no detectable levels were found in normal tissues. Sera from apparently normal patients contained a mean level of 2.2 units per ml of TAG-72. A level of 3 standard deviations above the mean level of TAG-72 found in normals was employed as a cut-off value to indicate a positive test result in subsequent studies. No patient with inflammatory disease or other benign colon disease exhibited elevated levels of TAG-72. Seven out of 20 patients with other carcinomas had elevated serum levels of TAG-72. The serum TAG-72 levels were compared with the serum levels of antigens recognized by the MAbs currently used to screen sera of carcinoma patients (CEA, GICA, and OC125). It was clearly demonstrated that TAG-72 is different from these antigens and can be found in some sera in which no antigen is detected by otherwise available MAb RIAs.