Estrogens play an important role in the brain function acting through two receptor types, ERα and ERβ, both well-recognized as transcription factors. In this study, we investigated the ERβ mRNA and protein levels in the rat hippocampus by using two in vivo models that are known to affect synapse formation. Natural estrous-proestrous cycle was used as a model in which a marked decrease in the density of hippocampal synapses was previously observed between proestrus and estrus. We have found that ERβ mRNA and protein were displayed in high levels in the estrus and in low levels in the proestrous phase. By applying kainic acid (KA) to adult rats, we demonstrated that up-regulation of ERβ mRNA and protein in hippocampal CA regions was vulnerable to KA-induced excitotoxicity. Furthermore, we note a concomitant decrease of ERβ in the excitotoxicity-resistant denate gyrus that undergoes intense plastic changes, including synaptogenesis. These data suggested that decreases in ERβ expression correlated with increase in synapse formation. This notion has been tested in vitro in hippocampal cultures, in which overexpression of ERβ by means of gene transfection resulted in the lowering of the dendritic spine density that was elevated by estrogen. In summary, our results suggest that ERβ inhibits synapse formation in hippocampal neurons.