The mouse estrogen receptor (mER) has been shown to exhibit stereospecific binding of certain stilbene estrogen agonists. The region of the mER involved in the stereochemical recognition of ligands was further defined using a stilbene isomer, Indenestrol B (IB). The IB compound has a chiral carbon bearing an ethyl substituent, and the wild type uterine mER has been shown to bind the enantiomers, IB-S and IB-R, with similar affinity. The wild type mER expressed in yeast exhibited a very minor preference for IB-S in transactivation (1.5-fold lower half-maximal dose than IB-R). The IB enantiomers could then be used to determine whether stereochemically distinct compounds with similar transcriptional activity utilize different amino acids in AF-2 for transactivation. Mutant mERs with glycine substitutions at Met521, His528, Met532, and Val537 were expressed in yeast and measured for IB-S- and IB-R-induced transactivation and ligand binding. The M532G mER showed a 124-fold and 50-fold reduction in transactivation induced by IB-S and IB-R, respectively, without a corresponding change in their ligand-binding affinities. Therefore, Met532 is required for transactivation induced by both IB enantiomers but does not discriminate based on stereospecificity. In contrast, the H528G mER displayed a gross change in stereospecific ligand recognition as illustrated by a 110-fold reduction in transactivation by IB-S and only a 8.8-fold decrease in activity by IB-R. As a result, H528G mER displayed a switch in ligand preference such that IB-R was now 8-fold more active than IB-S in transactivation. Therefore, His528 appears largely involved in transactivation specifically induced by IB-S but has a minimal influence in IB-S ligand binding. The remaining mutant mERs displayed wild type ligand binding and transactivation properties for the IB enantiomers illustrating no stereospecific recognition. These results imply that individual IB enantiomers bind to the mER with similar affinity but utilize at least one different amino acid within the AF-2 domain for signal transduction. The binding of stereochemically distinct ligands may alter the tertiary structure of the mER and cause repositioning of the AF-2 region that mediates transcription of specific genes and/or affect the binding of receptor-associated proteins, such as coactivators, which could influence transcription.