Neurokinin B (NKB) and its cognate receptor neurokinin 3 (NK3R) play a critical role in reproduction. NKB and NK3R are coexpressed with dynorphin (Dyn) and kisspeptin (Kiss1) genes in neurons of the arcuate nucleus (Arc). However, the mechanisms of action of NKB as a cotransmitter with kisspeptin and dynorphin remain poorly understood. We explored the role of NKB in the control of LH secretion in the female rat as follows. 1) We examined the effect of an NKB agonist (senktide, 600 pmol, administered into the lateral cerebral ventricle) on luteinizing hormone (LH) secretion. In the presence of physiological levels of estradiol (E 2), senktide induced a profound increase in serum levels of LH and a 10-fold increase in the number of Kiss1 neurons expressing c-fos in the Arc (P Ͻ 0.01 for both). 2) We mapped the distribution of NKB and NK3R mRNAs in the central forebrain and found that both are widely expressed, with intense expression in several hypothalamic nuclei that control reproduction, including the Arc. 3) We studied the effect of E 2 on the expression of NKB and NK3R mRNAs in the Arc and found that E 2 inhibits the expression of both genes (P Ͻ 0.01) and that the expression of NKB and NK3R reaches its nadir on the afternoon of proestrus (when circulating levels of E 2 are high). These observations suggest that NKB/NK3R signaling in Kiss1/NKB/ Dyn-producing neurons in the Arc has a pivotal role in the control of gonadotropin-releasing hormone (GnRH)/LH secretion and its regulation by E 2-dependent negative feedback in the rat. estradiol; hypothalamus NEUROKININ B (NKB) is a member of the tachykinin family that has recently emerged as a key neuropeptide in the control of reproductive function. Humans bearing loss-of-function mutations of TAC3 or TAC3R, which are homologous to NKB and its cognate receptor neurokinin 3 (NK3R, aka Tac2 and Tac3r), respectively, in rodents, exhibit hypogonadotropic hypogonadism and infertility (34,38). Clues regarding NKB's specific role in the regulation of reproduction come from studies in animals. In rodents, the compensatory rise of luteinizing hormone (LH) following ovariectomy (OVX) is ablated by treatment with the NKB agonist senktide (24,30). In addition, the expression of NKB and its receptor NK3R in the hypothalamic arcuate nucleus (Arc) is inhibited by estradiol (E 2 ) via estrogen receptor-␣ (9, 24). Together, these observations suggest that NKB/NK3R signaling plays an important role in the E 2 -dependent negative-feedback control of gonadotropin-releasing hormone (GnRH) and LH in mammals.In the Arc of sheep and rodents, NKB is coexpressed with kisspeptin and dynorphin, which are encoded by the Kiss1 and preprodynorphin (Dyn) genes, respectively (4, 15, 24). Kisspeptin is a potent GnRH secretagogue (25), and GnRH neurons express the kisspeptin receptor (Kiss1r, aka GPR54) (17), which plays a critical role in the neuroendocrine regulation of GnRH and LH secretion (8,25,31). Kisspeptin neurons in the Arc express estrogen receptor-␣ and are thought to be direct targets f...