|Abstract||Epidermal growth factor (EGF) and estrogen are potent regulators of breast tumorigenesis. Their short-term actions on human breast epithelial cells have been investigated extensively. However, the consequence of a long-term exposure to EGF and estrogen remains to be fully elucidated. The present study examined the effects of long-term exposure to EGF and 17?-estradiol on the proliferation, transformation, expression of markers of stemness, and tumorigenesis of McF7 human breast adenocarcinoma cells. Exposure to EGF and/or 17?-estradiol irreversibly enhanced the proliferation rate of McF7 cells, even following withdrawal. However, in a mouse xenograft experiment, no significant difference in tumor volume was observed between tumors derived from cells exposed to EGF, 17?-estradiol or EGF + 17?-estradiol. Immunohistochemistry performed on tumors derived from 17?-estradiol-exposed cells revealed reduced cell proliferation and vessel scores, according to the results obtained using Ki67 and von Willebrand factor staining, respectively. The EGFand/or 17?-estradiol-treated cells exhibited an increased ratio of cluster of differentiation (cd)44+/cd24 cells and enhanced ability to form mammospheres. Furthermore, the long-term exposure of McF7 cells to EGF and 17?-estradiol altered their responsiveness to short-term stimulatory or inhibitory treatments with EGF, 17?-estradiol, transforming growth factor-?1 (TGF?1), Iressa and SB431542. Therefore, the findings indicated that sustained exposure of McF7 cells to EGF and/or 17?-estradiol resulted in enhanced cell proliferation and mammosphere formation, an increased ratio of cd44+/cd24 cells, and altered responses to short-term treatments with EGF, 17?-estradiol, TGF?1, and drugs inhibiting these signaling pathways. However, this sustained exposure was not sufficient to affect tumor take or volume in a xenograft mouse model.