Current classification of breast cancers depends in great part on the expression of human epidermal growth factor receptor 2 (HER2), a cell surface tyrosine kinase receptor, and estrogen receptor (ER), the nuclear receptor for estrogen. In addition to reliable biomarkers, these receptors are targets of effective and widely used antitumor drugs. During malignant progression, HER2 and ER can establish an intricate cross-talk. In some cases, HER2 overexpression leads to the downregulation of ER and undermining of anti-ER therapies. A subgroup of HER2-positive breast cancer patients with poor prognosis expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTF) collectively known as p95HER2. One of these fragments, 611-CTF, is oncogenic in a variety of preclinical models. However, because of the lack of an appropriate tool to specifically analyze its levels in the clinical setting, the value of 611-CTF as a biomarker has not been established yet. Here, we show that 611-CTF induces resistance to anti-estrogen therapy and a more pronounced down-modulation of ER than that induced by full-length HER2. To validate this effect in breast cancer samples, we developed specific anti-611-CTF antibodies. With these antibodies, we showed that, whereas the frequency of ER positivity in HER2-positive/611-CTF- negative tumors (72.6%) is similar to that reported for HER2-negative tumors (70-80%), the number of ER-positive tumors in the 611-CTF-positive subgroup is very low (31.2%). These results reveal a mechanism of ER regulation mediated by HER2, which suggests a new strategy to improve responses to endocrine therapy in breast cancer. ©2010 AACR.