Heterologous specific antisera against human mammary epithelial antigens (HME-Ags), which are present in the human milk fat globule membrane and breast epithelial cells, were used in a solid-phase radioimmunoassay to determine the presence of these antigens in the sera of patients with disseminated cancer of the breast and other organs. Breast cancer patients carry high levels of HME-Ags in their circulation, while patients with disseminated nonbreast cancer, as well as normal female controls, do not. A similar release of HME-Ags in the circulation was shown by us in a model system. To further corroborate these findings, a three-step procedure for the extraction and identification of HME-Ags from the sera was devised. In this analytical procedure, circulating HME-Ags are recovered on a solid phase carrying their corresponding antibody (anti-HME) and radioiodinated in situ. Later, the labeled HME-Ags are released from the solid phase and characterized by NaDodSO4 gel electrophoresis. With this procedure, HME-Ags were isolated from sera of breast cancer patients but not from sera of nonbreast cancer patients or of normal female controls. The extracted HME-Ags had molecular masses of 150,000, 70,000, and 46,000 daltons. To further support these findings, a monoclonal antibody, BLMRL-HMFG-Mc3, directed to the 46,000-dalton HME-Ag was also used to extract its corresponding antigen from sera. Breast cancer patient sera contained such antigen while the sera of the other patients and controls did not. This highly sensitive methodology offers a specific approach to breast cancer diagnosis as well as further insight into the nature of circulating antigens with a view to increasing our understanding of breast cancer biology.