We determined cytosol c-erbB-2 protein levels using a sandwich enzyme immunoassay in benign breast disease and primary and recurrent breast cancer and analyzed the relationship between c-erbB-2 protein levels and clinicopathological factors. Overexpression of c-erbB-2 protein, the cut-off value being set at 18 ng/mg protein, was observed in 26 of the 139 cases of stages I-IIIB breast cancer (18.7%), four of the 12 cases of stage IV breast cancer (33.3%) and seven of the 13 recurrent breast cancer cases (53.8%). The levels of c-erbB-2 protein were significantly different between the stages. Overexpression of c-erbB-2 protein in stages I-IIIB breast cancer was associated with histological grade and serum CEA level, but not with other clinicopathological factors. In addition, there was an inverse correlation in the group of stages I-III plus IV breast cancer between c-erbB-2 protein expression and estrogen receptor status. Overexpression of c-erbB-2 protein can be easily determined in the cytosol fraction together with hormonal receptor by this method. The prognostic importance will be evaluated in ongoing adjuvant trials for operable breast cancer patients.