ERα-negative breast tumors tend to overexpress growth factor receptors such as epidermal growth factor receptor or c-erbB-2. Raf-1 is a key intermediate in the signal transduction pathways of these receptors. High levels of constitutive Raf kinase (Δraf) activity imparts ERα- positive MCF-7 breast cancer cells with the ability to grow in the absence of estrogen. Δraf transfectants maintained in estrogen-depleted media showed greatly diminished responses to 17β-estradiol or the pure antiestrogen ICI 182,780. Western blotting, ligand binding, and immunohistochemistry assays revealed a loss of ERα protein expression, and ribonuclease protection assays indicated that this correlated with loss of ERα message. In examining the basal expression of estrogen-induced genes in the stable transfectants or in transient cotransfection assays with an estrogen-response element-reporter construct and Δraf or constitutively active MAPK kinase (ΔMEK), no ligand- independent activation of ERα was observed. Transient expression of Δraf and double-label immunostaining showed ERα was lost in those cells that transiently expressed Δraf. Abrogation of Raf signaling via treatment with the MEK inhibitors PD 098059 or U0126 resulted in reexpression of ERα. Similar studies performed with MCF-7 cells overexpressing epidermal growth factor receptor or c-erbB-2 confirmed that hyperactivation of MAPK resulted in down-regulation of ERα that was reversible by MEK inhibition or transfection with dominant negative ERK1 and ERK2 constructs. These data suggest that the hyperactivation of MAPK in epidermal growth factor receptor- or c-erbB-2-overexpressing breast cancer cells is directly responsible for generation of an ERα-negative phenotype and, more importantly, that this process may be abrogated by inhibiting these pathways, thus restoring ERα expression.