This paper describes the establishment of an antiestrogen-resistant MCF7 breast cancer cell subline (FASMCF) by continuous culture of the estrogen-responsive parental line in steroid-depleted, ICI 182,780 (Faslodex; 10(-7) M)-supplemented medium. After a 3-month period of growth suppression, cells began to proliferate in ICI 182,780 at rates similar to those of untreated wild-type cells. Immunocytochemistry showed these cells to have reduced estrogen receptor and an absence of progesterone receptor proteins. RT-PCR and transient transfection studies with estrogen response element-reporter constructs confirmed that ICI 182,780-suppressed estrogen response element-mediated signaling. FASMCF cells show increased dependence upon epidermal growth factor receptor (EgfR)/mitogen-activated protein kinase (MAPK)-mediated signaling. Thus, EgfR protein and messenger RNA, growth responses to transforming growth factor-alpha, and extracellular signal-regulated kinase 1/2 MAPK activation levels are all increased. Unlike wild-type cells, FASMCF cells are highly sensitive to growth inhibition by an EgfR-specific tyrosine-kinase inhibitor (TKI), ZD1839 (Iressa), and an inhibitor of the activation of MEK1 (MAPKK), PD098059. Short-term ( approximately 3 weeks) withdrawal of cells from antiestrogen had no effect on growth or phenotype, whereas longer withdrawal (>10 weeks) appeared to partially reverse the cellular phenotype with increasing estrogen receptor and decreasing EgfR levels. In subsequent studies FASMCF cells were maintained in TKI, where their growth was again suppressed and secondary TKI resistance failed to develop within the 3-month period in which initial ICI 182,780 resistance arose. Furthermore, wild-type cells similarly maintained in combination ICI 182,780 and TKI treatment conditions remained growth arrested (>6 months), with notable cell loss through both reduced rates of cellular proliferation and increased cell death.