The ventromedial hypothalamus (VMH) is a sexually dimorphic region of the brain related to female reproductive behavior. The effect of estrogen in the adult rat VMH is thought to be mediated predominantly via estrogen receptor (ER)alpha, because this receptor is expressed at considerably higher levels than ER beta. The present study revealed, using in situ hybridization and immunohistochemistry, that both ER beta mRNA and protein were expressed in the ventrolateral portion of the caudal VMH, at remarkably higher levels during early postnatal development than in adulthood. In addition, the expression was sexually dimorphic, with females having significantly more ER beta-immunoreactive (-ir) cells than males, between postnatal d 5 (P5) and P14, although the sex difference was not significant by P21. Double-label immunofluorescence revealed that 66% of ER beta-ir cells coexpressed ER alpha in the caudal VMH of the P5 female rat. Furthermore, neonatal treatment with E2 benzoate down-regulated ER beta mRNA in the female rat VMH at P5 and decreased VMH ER beta-ir cells during the period between P5 and P14. In contrast to females, no differences in expression of ER beta mRNA or protein were detected between control and E2 benzoate-treated males. These results suggest that estrogen is involved in regulating the sexually dimorphic expression of ER beta in the VMH during early postnatal development of the rat.