Clinical studies including thousands of breast cancer patients have shown that c-erbB-2 is amplified and overexpressed in 20-30% of invasive human breast cancers and that it is associated with distant metastasis in specified patient subgroups. To isolate and characterize hematogeneously spreading c-erbB-2-positive epithelium-derived cells from the peripheral blood of breast cancer patients, a combined buoyant density gradient and immuno-magnetic separation method has been used. The method utilizes a biotinylated anti-cytokeratin monoclonal antibody (MAb) for capturing the epithelium-derived cells. The expression of c-erbB-2 by the captured cells was detected using an anti-c-erbB-2 rabbit antibody (21N) coupled to an anti-rabbit gold-labeled antibody, whereby immunoenzymatic cytokeratin staining was performed using a silver-enhanced immunogold double staining protocol. In total, 29 of the 46 patients tested had either cytokeratin (24/29) or cytokeratin/c-erbB-2 (19/29) positive clustered cells in their peripheral blood. We thus report here the presence and the frequency of clone-specifically stained clustered cells in the peripheral blood of breast cancer patients. The frequency of cytokeratin/c-erbB-2 doublepositive clustered cells in the peripheral blood was on average 10 times higher than that of double-positive single cells. The numbers of cytokeratin/c-erbB-2 double-positive clustered cells were positively correlated with the stage of tumors. Results of in vitro motility experiments using single and clustered cells from primary breast cancer tissue strongly support the assumption that cytokeratin/c-erbB-2 doublepositive clustered cells have a high potential for locomotion. We suggest that blood-borne epithelium-derived c-erbB-2positive clustered cells are the possible precursor cells responsible for the formation of distant metastases and bone marrow micrometastases.