The c-erbB-2 oncogene is frequently amplified and overexpressed in human breast cancer. We have studied the c-erbB-2 protein in conjunction with DNA content in frozen samples from breast cancers using flow cytometry. The cell suspensions were obtained by mechanical disaggregation followed by a short fixation in 1% paraformaldehyde. The level of c-erbB-2 expression was calculated as a fluorescence index, taking into account the relative amount of total cellular fluorescence compared to nonspecific fluorescence. The flow cytometric value correlated with immunohistochemical results obtained with the same monoclonal antibody (c-neu, clone 9G6). Overexpression of c-erbB-2 was significantly more frequent in DNA aneuploid tumors than in DNA diploid ones and correlated with increasing S-phase fraction and estrogen receptor negativity. In 10 DNA multiploid tumors, the different aneuploid stemlines uniformly expressed c-erbB-2, supporting the hypothesis that overexpression is an early event in breast cancer. Of the 172 tumors, the 37 (22%) judged as positive with immunohistochemistry showed a somewhat higher rate of distant recurrence than others (P = 0.14). The fluorescence index was significantly associated with prognosis (P = 0.0012), as it was also among the immunohistochemically positive cases. If the degree of overexpression is important, then flow cytometry could be a feasible technique for classification.