Fertility in humans and animals is driven by the pulsatile secretion of gonadotrophin-releasing hormone (GnRH) neurons in the hypothalamus. The negative and positive feedback effects of estradiol modulate it, but indirectly since GnRH neurons do not express estrogen receptors. RFamide-related peptide-3 (RFRP-3), a neuropeptide produced by neurons with cell bodies distributed around the dorsomedial hypothalamus, has been postulated to act upstream of GnRH to modulate the negative feedback effects of estradiol, and to inhibit the surge of GnRH and gonadotrophins that triggers ovulation. Many RFRP-3 neurons express estrogen receptor-a (ERa), project to GnRH neurons and suppress their activity. The aim of the current study was to investigate the role of RFRP-3 in mediating the control of fertility by estradiol. We first generated transgenic mice with ERa deleted specifically from RFRP-3 neurons using Cre-Lox transgenics approach. Onset of puberty, estrous cyclicity, bodyweight and fertility were measured in knockout (KO) mice and their control littermates. We found that female mice with ERa deleted from RFRP-3 neurons exhibited a significant 3.24 + 0.18-day advance in puberty onset (p<0.05), and disordered subsequent estrous cycles. In contrast, we found that the male mice exhibited a 4.45 + 0.22-day delay in puberty onset (p<0.05). In addition, the male KO mice sired a 13% smaller litter size (p<0.05) and a 24% reduced number of litters (p<0.05) over a 126 day breeding study compared to the controls. Measurement of fertility in female mice is currently in progress. However ovariectomised and estradiol-treated KO females displayed an impaired estradiol negative and positive feedback compared to the controls. It is possible that the latter effect represents a secondary response to impaired negative feedback prior to the surge. These results highlight the importance of RFRP-3 neurons as key players in the hypothalamic control of fertility via estradiol.