Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways via protein-protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E(2)), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein-protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E(2))- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen-ERα complexes in human BC cell nuclei. In particular, the E(2)-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds.