A novel technique in multiparameter flow cytometry (FCM) using dual laser excitation of three fluorescent dyes has been developed to differentiate breast epithelial cells from stromal components. This technique has been applied to determine the expression of the oncoprotein c-erbB-2 in breast epithelial tumors. SK-BR-3, a breast cancer cell line with c-erbB-2 overexpression, can be identified by FCM from a mixed cell suspension using a monoclonal anti-human cytokeratin antibody conjugated with fluorescein isothiocyanate. Using the mouse monoclonal anti-c-erbB-2 antibody, TA-1 (4.8 +/- 1.0 x isotype control), the c-erbB-2 oncoprotein overexpression in breast epithelial cells can be detected as an increase in indirect blue fluorescence by aminomethyl coumarin. MCF-7, a breast cancer cell line with normal c-erbB-2 expression, has baseline blue fluorescence (1.0 +/- 0.5 x isotope control). Twenty-one fresh breast specimens have been examined by FCM. Overexpression of c-erbB-2, defined by blue fluorescence ratio of TA-1/isotype control > or = 1.5 (> 3 SD from baseline), is detected in 0 of 3 patients with Stage I cancer, 5 of 14 patients with Stage II and III cancer, and 3 of 4 patients with proliferative disease. Patients with elevation of oncoprotein detected by FCM have corresponding RNA overexpression detected by Northern blot hybridization and increased gene amplification detected by Southern blot hybridization. FCM allows for the simultaneous identification of breast epithelial cells and the selective examination of these cells for the expression of c-erbB-2 oncoprotein, thus minimizing stromal contamination. This represents a novel application of FCM with potential for wide clinical applicability.