The candidate tumor-specific soluble mammary tumor glycoprotein with a molecular weight of approximately 20,000 (MTGP20) has been isolated from human breast carcinomas and characterized biochemically. Although with the use of xenoantisera this glycoprotein has previously been demonstrated only in breast carcinomas, analyses of body fluids and conclusions regarding putative tumor specificity have been limited by the sensitivity of assays. In the present study, a specific radioimmunoassay has been developed. With appropriate selection of buffer, the assay has a sensitivity threshold of less than 0.1 unit of MTGP20 per ml, equivalent to less than 250 pg type I MTGP20 per ml or less than 530 pg type II MTGP20 per ml, which is more than 200-fold more sensitive than previously described assays. MTGP20 type I, which contains tyrosine, was labeled with 125I and used as the immunochemical ligand in a double antibody competitive inhibition assay. A partial weak cross-reaction was observed with a mixture of placental glycoprotein (perchloric acid extract), but this reaction was abolished by absorption of antisera with placental glycoprotein. Normal tissue and tumors of other than breast origin were devoid of MTGP20-related antigens. MTGP20-related antigens were not detectable in sera or concentrated urine specimens from normal individuals or patients with metastatic breast carcinomas. The present studies further support the probable tumor specificity of this glycoprotein but indicate that it does not represent a circulating secretory product of the cancer cell and does not provide a serum marker for breast carcinoma.