Estrogen receptors (ER and ER ) are ligand-activated nuclear receptors that mediate the action of estrogens. These receptors activate transcription by similar mechanism(s), although the overall amino acid sequence identity is only 47%. In order to compare the structural and conformational features of ER and ER , we monitored their intrinsic tryptophan fluorescence during thermal unfolding. The 50% unfolding temperatures (T M ) of ER and ER were 39±1 and 40±2°C, respectively. Estradiol had no significant effect on the T M of ER or ER . In contrast, binding of the estrogen-response element increased the T M of ER and ER by 10°C. Thermal unfolding of estradiol-bound ER and ligand-free ER showed two-step transitions, with the formation of intermediates that were stable between 36-48 and 34-42°C, respectively. We confirmed the presence of intermediate states during thermal unfolding by circular dichroism spectroscopy. Atomic force microscopy showed that the ER intermediate consisted of discrete globular particles, whereas the ER intermediate showed a speckled appearance, with sparse well-defined particles. Fluorescence-quenching studies showed the presence of two classes of tryptophan in unliganded ER and ER . Binding of estradiol to ER exposed its tryptophans, whereas estradiol reduced the accessibility of the tryptophans of ER . Our results illustrate the differential effects of ligands on the unfolding of ER and ER , and identify partially unfolded intermediates. Differences in the conformational flexibility and stability of ER and ER may represent functional differences of ligand-bound ERs in recruiting coactivator proteins and initiating transcription.