Our previous studies with estrogen receptor  knockout (ER ؊/؊ ) mice demonstrated that ER 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 ER in the developing brain and identified a population of ER-regulated interneurons. ER 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, ER expression increased in the hypothalamus, thalamus, and midbrain and appeared in the limbic forebrain. At E18.5, ER 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 ER. In ER ؊/؊ 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 ER ؊/؊ than in WT mice at E15.5 and, unlike WT mice, was absent from the superficial marginal zone. These findings suggest that ER in the embryonic brain is necessary for the development of calretininimmunoreactive GABAergic interneurons and for neuronal migration in the cortex through modulating epidermal growth factor receptor expression at middle and later embryonic stages.cortex ͉ EGF receptor ͉ striatum ͉ thalamus E strogen is important during brain development, influencing the maturation of neural systems and affecting the sexual differentiation of brain structures and functions (1, 2). Sexual differentiation in the brain occurs in male rodents at day 18.5 when aromatase is induced in the brain and catalyses the synthesis of estradiol from testosterone secreted from the embryonic testis. At this time, estrogen imprints the neuroendocrine control of gonadal function, reproductive behavior, and the sexual differences in the secretion of growth hormone.Estrogen actions now are recognized to occur via two distinct estrogen receptors (ER), ER␣ and ER, that reside in cell nuclei (3, 4). In the brain, ER␣ plays a critical role in regulating reproductive neuroendocrine function, whereas ER may be more important in regulating nonreproductive functions (5-8).In ER␣ knockout mice, no gross brain abnormalities have been reported, whereas ER plays a prominent role during embryogenesis and there are significant morphological abnormalities in the embryonic brains of ER knockout (ER Ϫ/Ϫ ) mice (9). Studies have revealed that ER also is expressed in fetal human brain and may mediate effects of estrogen in the developing nervous system (10). Lemmen et al. (11) reported that ER mRNA is detectable in brains of mouse embryo at embryonic day 10.5 (E10.5), but there is no i...