A significant number of HER-2 amplified breast cancers is effectively treated by trastuzumab and further shows receptor-enhanced chemosensitivity. Recent studies have postulated transactivation of HER-2 also in tumors expressing phosphorylated/activated HER-2 (pHER-2) and of the HER-3/HER-4 ligand heregulin (HRG), independent of HER-2 amplification. As a consequence, a subset of tumors without HER-2 overexpression would be sensitive to trastuzumab chemotherapy. To investigate the potential transactivation of HER-2, in 171 breast cancers from the GENICA study with negative/low expression of HER-2 we analyzed the expression of pHER-2, HRG, HER-3 and HER-4 by immunohistochemistry. None of the tumors examined displayed expression of pHER-2. Moderate or strong cytoplasmic staining of HRG, HER-3 and HER-4 was observed in 44 (26%), 67 (39%) and 33 (19%) cases, respectively. No association of HRG, HER-3 and HER-4 with the survival of patients or with known prognostic clinical factors was seen. In conclusion, our data obtained on a well-characterized cohort of breast cancers provide no evidence of HER-2-activation in the absence of HER-2 overexpression. The biological function and clinical implications of HRG, HER-3 and HER-4 in this group of tumors remain unclear. Our results cannot support the hypothesis of a transactivation of HER-2 and thus a possible therapeutic benefit of trastuzumab in HER-2 negative breast cancers.