Estrogen has diverse roles in the regulation of both reproductive and non-reproductive functions. In target tissues including the brain, these actions of estrogen are mediated mainly via estrogen receptor (ER), ER-α and ER-β. Many of estrogenic actions change with age, leading to various pathological alterations during the postmenopausal stage. One reason for the changes in estrogenic actions is the age-dependent decrease in circulating level of estrogen itself. Another possible reason is the reduced sensitivity to estrogen with age. Concerning ER-α, many previous studies using aged female rodents have reported that the effects of estrogen treatment on ER-α expression in the brain alter with age. On the other hand, little is known about the effects of estrogen on ER-β expression in aged female brain. In the present study, to reveal the effect of aging on estrogen-dependent changes in ER-β mRNA expression in female rat brain, we performed in situ hybridization using young and middle-aged female rats, and examined the number of ER-β mRNA-positive cells throughout the brain. All rats were ovariectomized bilaterally, and then implanted with a Silastic capsule containing 17β-estradiol or an empty capsule. In young rats, estrogen treatment decreased the number of ER-β mRNA-positive cells in the external plexiform layer of the olfactory bulb, entorhinal cortex, intermediate part of the lateral septal nucleus, nucleus of the horizontal limb of the diagonal band, amygdala (lateral, medial, and basolateral nuclei), thalamic nuclei, medial geniculate nucleus, suprachiasmatic nucleus, and Purkinje cells in the cerebellum. In middle-aged rats, estrogen treatment decreased the number of ER-β mRNA-positive cells in the mitral cell and external plexiform layers of the olfactory bulb, central amygdaloid nucleus, medial geniculate nucleus, posterior hypothalamic nucleus, suprachiasmatic nucleus, and reticular part of the substantia nigra. Our results showed that the regions in which expression of ER-β mRNA expression is affected by estrogen in middle-aged females differ from those in young females, suggesting the effects of estrogen on ER-β expression change with age.