We have previously demonstrated that ERβ is necessary for embryonic development of the brain as early as E14.5 and is involved in neuronal migration. Such early effects of estrogen receptors were unexpected because estradiol synthesis and action in the brain are well documented to occur at E18.5.Since the presence of ERβ in the embryonic brain has not been demonstrated, it remained possible that the developmental abnormalities in ERβ−/− mice were due to indirect effects of estrogen action on the brain. In the present study we examined the distribution of ERβ in the developing brain and identified a population of ERβ-regulated interneurons ERβ appears in the brain at E12.5, mainly localized in the wall of midbrain, neuromere, hypothalamus, thalamus and basal plate of pons. At E15.5 and E16.5, ERβ expression increased in the hypothalamus, thalamus and midbrain and appeared in the limbic forebrain. At E18.5, ERβ expression was strongly and widely expressed throughout the brain including cerebellum and striatum, while there were very few positive cells in the ventricular region. In the paraventricular thalamic nucleus and parafascicular nucleus, most of the calretinin-immunopositive interneurons expressed ERβ. In ERβ−/− mice, calretinin expression was markedly lower than in WT mice in the hippocampus, thalamus and amygdala both at E16.5 and at E18.5.These findings suggest that ERβ is necessary for the development of calretinin-immunoreactive GABAergic interneurons and for neuronal migration in the cortex.