Estrogen plays an important role in sexual differentiation of the brain in rats during the perinatal period. To elucidate molecular mechanisms underlying sexual differentiation of the brain, in this study we investigated genes differentially expressed between sexes or induced to express by estrogen in neonatal rat hypothalamus using DNA microarray analysis in combination with real-time RT-PCR. It was found that the levels of expression of the genes encoding glutamic acid decarboxylase 65 and coronin 1b were higher in male than female hypothalamus on postnatal day (PN) 5 and those of collagen type 3 α1 and thioredoxin reductase 2 genes in female hypothalamus on PN5 were decreased and increased, respectively, by treatment with estradiol on PN2. Then the developmental changes in the expression of these 4 genes were examined from 1 day before the parturition to PN9, and they all showed sexual dimorphic patterns. In addition, dependence of the expression of these genes on either estradiol, testosterone or dihydrotestosterone during the neonatal period was confirmed. These results suggest that these four genes are involved in sexual differentiation of the rat brain, and that androgen per se as well as estrogen may take part in the processes.