Since the poor prognosis associated with HER2 amplified breast cancers might be explained by a mechanistic association between p185^HER2 overexpression and therapeutic resistance, we assessed the chemo-endocrine sensitivity of estrogen receptor (ER) containing MCF-7 breast cancer cells transfected with full-length HER2 cDNA. Of the 36 isolated MCF/HER2 subclones, 7 were found to overexpress p185^HER2 surface receptor at levels 3 to 45-fold greater than parental or control transfected cells (MCF/neo). The overexpressing transfectants possessed increased inositol-1,4,5-trisphosphate-3'-kinase activity comparable to enzyme activity in the endogenously HER2 amplified breast cancer cell lines SK-Br-3 and BT-474. The anti-p185^HER2 monoclonal antibody and receptor-specific partial agonist, muMAb4D5 (4D5), known to inhibit growth of SK-Br-3 and BT-474 cells, produced no significant growth inhibitory effect on any of the transfectants including the 45-fold overexpressing MCF/HER2–18 cells which were studied in greater detail. MCF/HER2–18 cells contained at least partially functioning exogenous receptor since 4D5 (3µg/ml) specifically stimulated phosphorylation of p185^HER2 and its co-precipitating ptyr56 substrate within 5 min, and this was followed at 1 h by a transient induction ofc-myc but notc-fos mRNA. ER content and thein vitro sensitivity of MCF/HER2–18 cells to 5-fluorouracil and adriamycin were identical to those of control transfectants and parental cells. However, these highly overexpressing transfectants had acquired low level (2 to 4-fold) resistance to cisplatin and were no longer sensitive to the antiestrogen tamoxifen (TAM). To compare the hormone-dependent tumorigenicity of the HER2 transfectants, MCF/HER2–18 and control cells (MCF, MCF/neo-3) were implanted into ovariectomized athymic nude mice. No tumors were produced in the absence of estradiol (E₂) administration. In E₂ supplemented mice, MCF/HER2–18 tumors grew most rapidly. When E₂ treatment was stopped and daily TAM injections were initiated, MCF-7 and MCF/neo-3 tumor growth ceased immediately, while MCF/HER2–18 tumors continued to show an accelerated growth rate lasting weeks. This pattern of hormone-dependent, TAM-resistant growth exhibited by the MCF/HER2–18 tumors in nude mice supports the possibility that p185^HER2 overexpression in human breast cancers may be linked to therapeutic resistance.