In most mammals, RF-amide-related peptides are synthesized in the dorsomedial hypothalamic nucleus and regulate reproduction via inhibiting GnRH neurons and, possibly, adenohypophyseal gonadotrophs. In the present study, we investigated the possibility that RFRP-synthesizing neurons are involved in estrogen feedback signaling to the reproductive axis in mice. First, we used quantitative in situ hybridization and compared the expression of prepro-RFRP mRNA of ovariectomized mice, with and without 17β-estradiol (E2) replacement. Subcutaneous administration of E2 via silastic capsules for 4 d significantly down-regulated prepro-RFRP mRNA expression. The underlying receptor mechanism was investigated with immunohistochemistry. In ovariectomized mice, low levels of nuclear estrogen receptor (ER)-α immunoreactivity were detectable in 18.7 ± 3.8% of RFRP neurons. The majority of RFRP neurons showed no ER-α signal, and RFRP neurons did not exhibit ER-β immunoreactivity. Results of these studies indicate that RFRP is a negatively estradiol-regulated neurotransmitter/neuromodulator in mice. The estrogenic down-regulation of RFRP expression may contribute to estrogen feedback to the reproductive axis. The issue of whether E2 regulates RFRP neurons directly or indirectly remains open given that ER-α immunoreactivity is present only at low levels in a subset of these cells.