Recent clinical evidence suggests that the neuroprotective and beneficial effects of hormone therapy may be limited by factors related to age and reproductive status. The patient's age and length of time without circulating ovarian hormones are likely to be key factors in the specific neurological outcomes of hormone therapy. However, the mechanisms underlying age-related changes in hormone efficacy have not been determined. We hypothesized that there are intrinsic changes in estrogen receptor β (ERβ) function that determine its ability to mediate the actions of 17β-estradiol (E2) in brain regions such as the ventral hippocampus. In this study, we identified and quantified a subset of ERβ protein interactions in the ventral hippocampus that were significantly altered by E2 replacement in young and aged animals, using two-dimensional differential gel electrophoresis coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. This study demonstrates quantitative changes in ERβ protein-protein interactions with E2 replacement that are dependent upon age in the ventral hippocampus and how these changes could alter processes such as transcriptional regulation. Thus, our data provide evidence that changes in ERβ protein interactions are a potential mechanism for age-related changes in E2 responsiveness in the brain after menopause.