We have previously reported the oncogenic properties of the gene amplified in breast cancer 1 (AIB1), a member of the p160 family of hormone receptor coactivators. In a transgenic mouse model, AIB1 overexpression resulted in a high incidence of tumors in various tissues, including mammary gland, uterus, lung, and pituitary. To determine whether the AIB1 oncogenicity in this model depended on its function as an estrogen receptor (ER) coactivator, we abolished ER signaling through two independent approaches, by performing ovariectomy on AIB1 transgenic (AIB1-tg) mice to prevent gonadal estrogen production and by crossing AIB1-tg mice with ERα-null mutant mice. Ovariectomized (ovx) mice, but not AIB1 × ERα−/− mice, still developed mammary gland hyperplasia and ductal carcinoma in situ. Both approaches, however, completely prevented the development of invasive mammary tumors, indicating that invasive mammary tumor formation is strictly estrogen dependent. Once developed, AIB1-induced mammary tumors can subsequently lose their dependence on estrogen: Injection of ERα(+) tumor cell lines derived from such tumors into ovx or untreated wild-type mice resulted in a similar rate of tumor growth in both groups. Surprisingly, however, ovx mice had an ∼4-fold higher rate of metastasis formation, suggesting that estrogen provided some protection from metastasis formation. Lastly, our experiments identified oncogenic functions of AIB1 that are independent of its ER coactivation, as both approaches, ovariectomy and ER−/− crosses, still resulted in a high incidence of tumors in the lung and pituitary. We therefore conclude that AIB1 can exert its oncogenicity through tissue-specific estrogen-dependent and estrogen-independent functions. Cancer Res; 70(10); 4102–11. ©2010 AACR.