Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17β-estradiol (E2) and the selective ER modulator raloxifene (RAL)] from the human ERα ligand-binding domain in monomeric and dimeric forms. E 2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II′). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I′). Remarkably, ER dimerization strongly suppresses Paths II and II′ for E2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family. Copyright © 2008 by The Endocrine Society.22715651578Aranda, A., Pascual, A., Nuclear hormone receptors and gene expression (2001) Physiol Rev, 81, pp. 1269-1304Jordan, V.C., Antiestrogens and selective estrogen receptor modulators as multifunctional medicines. 1. 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