Our previous studies with estrogen receptor beta knockout (ERbeta(-/-)) mice demonstrated that ERbeta is necessary for embryonic development of the brain as early as embryonic day 14.5 (E14.5) and is involved in neuronal migration. Such early effects of ER were unexpected because estradiol synthesis and action in the brain occur at E18.5. In the present study, we examined the distribution of ERbeta in the developing brain and identified a population of ERbeta-regulated interneurons. ERbeta appears in the brain at E12.5, mainly localized in the wall of the midbrain, neuromere, hypothalamus, thalamus, and basal plate of pons. At E15.5 and E16.5, ERbeta expression increased in the hypothalamus, thalamus, and midbrain and appeared in the limbic forebrain. At E18.5, ERbeta expression was strongly expressed throughout the brain, including the cerebellum and striatum, whereas 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 ERbeta. In ERbeta(-/-) mice, calretinin expression was markedly lower than in WT mice in the hippocampus, thalamus, and amygdala both at E16.5 and at E18.5. Epidermal growth factor receptor expression was lower in the cortex of ERbeta(-/-) than in WT mice at E15.5 and, unlike WT mice, was absent from the superficial marginal zone. These findings suggest that ERbeta in the embryonic brain is necessary for the development of calretinin-immunoreactive GABAergic interneurons and for neuronal migration in the cortex through modulating epidermal growth factor receptor expression at middle and later embryonic stages.