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Publisher Summary The estrogen receptor (ER) is a ligand-regulated transcription factor that belongs to the nuclear receptor (NR) superfamily and acts as a dimeric species. There are two subtypes of ER—ERα and ERβ—which are both mainly regulated by the endogenous estrogen—estradiol (E2). ER modulation is involved in the development and regulation of reproductive, cardiovascular, and bone health, in addition to controlling the various aspects of cognitive function. Ligands that bind to the ER can be categorized into three pharmacological classes: agonists, mixed agonist—antagonists, and pure antagonists. The mixed agonist–antagonists are also referred to as “selective estrogen receptor modulators (SERMs)” because of their tissue-selective agonist or antagonist activities. Nuclear receptors (NRs) form strong dimers that are essential for their functioning as transcription factors. NRs that bind steroid ligands (i.e., ER) typically function as dimers, either homodimers between identical receptor monomers or between closely related subtypes. NRs that bind nonsteroidal ligands, however, typically function as heterodimers with the retinoid X receptor (RXR). Using fluorescence resonance energy transfer (FRET) and fluorescent-labeled ERα-LBDs, the regulation of homodimer stability by ligands and coactivator peptides is monitored. This technique provides structure–activity relationships (SARs) of ligands in terms of ER dimer stability and can be used as a functional assay to identify the pharmacological character of novel synthetic or environmental ER ligands. Fluorescent-labeled ERβ-LBDs can be used to study homo- and heterodimers of both ER subtypes and the manner in which SERMs and ER subtype-selective ligands (both agonists and antagonists) modulate differential homo- and heterodimer stability. Site-specific fluorescent ERs can also be used for the development of other types of assays to characterize receptor conformation, conformational dynamics, and ligand or coregulator interactions.