Here we present the use of hydrogen-deuterium exchange (HDX) mass spectrometry in analyzing the estrogen receptor beta ligand binding domain (ERbeta LBD) in the absence and presence of a variety of chemical compounds with different binding modes and pharmacological properties. Previously, we reported the use of HDX as a method for predicting the tissue selectivity of ERalpha ligands. HDX profiles of ERalpha LBD in complex with ligand could differentiate compounds of the same chemotype. In contrast, similar analysis of ERbeta LBD showed correlation to the compound chemical structures but little correlation with compound tissue selectivity. The different HDX patterns observed for ERbeta LBD when compared to those for ERalpha LBD bound to the same chemical compounds serve as an indication that ERbeta LBD undergoes a different structural response to the same ligand when compared to ERalpha LBD. The conformational dynamics revealed by HDX for ERbeta LBD together with those for ERalpha LBD shed light on ER ligand interactions and offer new structural insights. The compound-specific perturbations in HDX kinetics observed for each of the two isoforms should aid the development of subtype-selective ER ligands.