Striking sex difference was detected in the expression of estrogen receptor (ER) beta mRNA and protein by nonisotopic in situ hybridization and immunohistochemistry in the anteroventral periventricular nucleus (AVPV) of the rat preoptic area. In females more than in males, a significantly larger number of ERbeta mRNA-positive cells were visualized in the medial-most portion of the AVPV within 50 microm from the ependymal lining of the third ventricle. Rats of 7, 14, 21, 35, and 60 days of age (d 1 = day of birth) showed the sex difference. Orchidectomy of male neonates or estrogen treatment of female pups reversed the brain phenotype when examined on d 14. In the AVPV of adult females, ERalpha immunoreactivity colocalized in 83% of ERbeta mRNA-positive cells. Tyrosine hydroxylase immunoreactivity colocalized in 18% of ERbeta immunoreactive cells in d 21 females. Infusion of an ERbeta antisense oligonucleotide into the third ventricle in the vicinity of the AVPV resulted in significantly longer days of successive estrus and a 50% reduction in the number of ERbeta-immunoreactive cells in the AVPV. These findings provide support for the hypothesis that activation of ERbeta in the AVPV is an important regulatory event in the female-typical induction of luteinizing hormone surge by estrogen.