Among hormone-inducible transcription factors, estrogen receptors (ERs) play important roles in tissue growth and differentiation, via either direct or indirect binding, in the nucleus, to specific DNA targets called estrogen responsive elements (EREs), or through nongenomic pathways. In humans, two estrogen receptor isoforms (hERs), designated hERα and hERβ, have been identified. These two hERs, encoded by genes located on distinct chromosomes, exhibit divergent tissue-specific functions and different subcellular distributions depending on their binding status, free or complexed to their cognate ligands. Because it is hypothesized that such distinct behaviors may arise from various conformational stabilities and flexibilities, the effect of salt concentration and temperature was studied on the free and estrogen-activated hERα and hERβ. Our results show that the conformational stability of hERβ is weakly modulated by salt concentration as opposed to hERα. In addition, we show that the estrogen-bound hERs exhibit a more constrained structure than the unliganded ones and that their conformational flexibility is more affected by diethylstilbestrol binding than that of estradiol, 4-hydroxytamoxifen, or raloxifen. In line with these results, conformational analysis and computational docking were performed on hERα and hERβ, which confer molecular support of a diethylstilbestrol-induced restrained flexibility as compared to other ligands. We found that Trp383 in hERα and Trp335 in hERβ can closely interact with the NR-box motif of the H12 helix and act as a gatekeeper of the agonist-bound versus antagonist-bound conformations. Altogether, our study contributes to an improved knowledge of the diverse physicochemical properties of full-length hERs, which will help in our understanding of their distinct cellular roles in various cellular contexts.