Estrogen receptor (ER) and human epidermal growth factor 2 (HER2) are well-investigated molecules and the focus of many breast cancer therapies. There is a group of breast cancers lacking ER and HER2, but it is not fully understood. Treatment for these patients is limited to cytotoxic chemotherapy. The purpose of present study is to examine ER(-)/HER2(-) breast cancers, with a particular focus on epidermal growth factor receptor (EGFR). EGFR is a target molecule for which novel medicines have been recently developed for other organ cancers, however biological significance in breast cancer is not yet well demonstrated. Breast cancer specimens (n=58) were categorized into four groups: i) ER(+)/HER2(-) (51.7%); ii) ER(+)/HER2(+) (8.6%); iii) ER(-)/HER2(+) (20.7%); and iv) ER(-)/HER2(-) (19.0%). They were immunohistochemically (IHC) examined using antibodies for EGFR, platelet derived growth factor receptor (PDGFR)alpha, PDGFRbeta, parathyroid hormone (PTH) receptor, Ki-67, cyclinD1, p53, and vimentin. The Ki-67 labeling index (LI) was highest in ER(-)/HER2(-) (36.5%), and decreased in order from ER(-)/HER2(+) (31.4%), ER(+)/HER2(+) (17.7%), to (ER(+)/HER2(-) (15.9%) (p=0.001). EGFR, p53 and vimentin were highly expressed in ER(-)/HER2(-) breast cancer cells (p<0.01). CyclinD1 was inversely expressed to Ki-67 LI (p<0.001). Gene amplification of EGFR was examined by two in situ hybridization techniques, fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) in serial sections to IHC. Only 1 of 14 EGFR-positive breast cancers showed gene amplification at low levels by CISH. Overall, the ER(-)/HER2(-) breast cancer showed the highest Ki-67 LI, the most frequent expression of EGFR, p53 and vimentin, as well as the lowest expression of cyclinD1. It is unlikely that gene amplification contributes to EGFR expression. ER(-)/HER2(-) breast cancers have potential in the development of novel therapeutics, including targeted medicines.