We have previously demonstrated that prolonged treatments with raloxifene (RAL) in vitro will result in phase II RAL resistance and RAL-induced tumor growth. Clinical interest prompted us to re-examine RAL resistance in vivo, particularly the effects of long-term treatments (a decade or more) on the evolution of RAL resistance. In this study, we have addressed the question of this being a reproducible phenomenon in wild-type estrogen receptor (ER)-positive human breast cell line MCF-7. MCF-7 cells cultured under estrogen-deprived conditions in the presence of 1 microM RAL for more than a year develop RAL resistance resulting in an independent cell line, MCF7-RAL. The MCF7-RAL cells grow in response to both estradiol E2 and RAL. Fulvestrant (FUL) blocks RAL and E2-mediated growth. Transplantation of MCF7-RAL cells into athymic ovariectomized mice and treatment with physiologic doses of E2 causes early E2-stimulated tumor growth. In contrast, continuous treatment of implanted animals with daily oral RAL (1.5 mg daily) causes growth of small tumors within 15 weeks. Continuous re-transplantation of the tumors growing in RAL-treated mice indicated that RAL stimulated tumor growth. Tumors in the untreated mice did not grow. Bi-transplantation of MCF7-E2 and MCF7-RAL tumors into the opposing mammary fat pads of the same ovariectomized animal demonstrated that MCF7-E2 grew with E2 stimulation and not with RAL. Conversely, MCF7-RAL tumors grew with RAL and not E2, a characteristic of phase II resistance. Established phase II resistance of MCF7-RAL tumors was confirmed following up to 7 years of serial transplantation in RAL-treated athymic mice. The ERalpha was retained in these tumors. The cyclical nature of RAL resistance was confirmed and extended during a 2-year evolution of the resistant phases of the MCF7-RAL tumors. The MCF7-RAL tumors that initially were inhibited by E2 grew in the presence of E2 and subsequently grew with either RAL or E2. RAL remained the major grow stimulus and RAL enhanced E2-stimulated growth. Subsequent transplantation of E2 stimulated tumors and evaluations of the actions of RAL, demonstrated robust E2-stimulated growth that was blocked by RAL. These are the characteristics of the anti-estrogenic actions of RAL on E2-stimulated breast cancer growth with a minor component of phase I RAL resistance. Continuous transplantation of the phase I RAL-stimulated tumors for >8 months causes reversion to phase II resistance. These data and literature reports of the cyclical nature of anti-androgen/androgen responsiveness of prostate cancer growth, illustrate the generality of the evolution of anti-hormonal resistance in sex steroid-sensitive target tissues.