During the last decade our understanding of the hypothalamic-pituitary-gonadal axis (HPG) has dramatically expanded. Kisspeptins are reported to be the most potent activators of HPG to date. In vivo and in vitro studies suggest that the kisspeptin/GPR54 system is very important to fertility control, and kisspeptin is the main triggering factor for puberty onset. A rise in intracellular free calcium concentration ([Ca2+]i) is the common trigger for exocytosis in most cell types. Several neuropeptides are known to regulate GnRH secretion from the hypothalamus by changing [Ca2+]i. Kisspeptins have been recently suggested to be the potent activator of GnRH neurons. Kisspeptin-10 causes a triphasic change in [Ca2+]i in GT1-7 cells (1). But, very little is known about regulation of this newly identified regulator of reproduction, kisspeptin. The novel hypothalamic RFamide peptides, avian gonadotropin-inhibitory hormone (GnIH) and its mammalian orthologous peptides, discovered in mammals belonging to the RF-amide peptide superfamily (RFRP) seem to be important regulators of the reproductive axis. Kisspeptin and its receptor (GPR54) are expressed in rHypo8 cells, which are immortalized kisspeptin neurons newly developed from rat hypothalamus (2). Whether GnIH/RFRP has modulatory effects on kisspeptin-activated rHypo8 cell line remains to be determined. Thus, in this study for the first time the effects of GnIH/RFRP-1 on calcium signaling in rHypo8 cells were investigated. rHypo8 cells were cultured on poly-l-lysine-coated coverslips and maintained in DMEM supplemented with heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 oC in 5% CO2. After loading the cells with 1 μmol Fura-2 AM, [Ca2+]i responses were quantified by the changes in 340/380 ratio for individual rHypo8 cells. Kisspeptin-10 at 100 nM caused a significant increase (p<0.001) in [Ca2+]i compared to basal levels in rHypo8 cells (n=58). GnIH at 1 µM (n=47) inhibited (p<0.001) kisspeptin-induced [Ca2+]i increase. RFRP-1 at 1 µM (n=72) did not have any significant effect on [Ca2+]i in rHypo8 cells. These results suggest that GnIH may exert its effects on HPG axis by modulating kisspeptin neurons.