Hypothalamic RFamide peptides, gonadotropin-inhibitory hormone (GnIH) and orthologous peptides, which were then discovered in mammals belonging to the RF-amide peptide superfamily (RFRP), seem to be important regulators of hypothalamus-pituitary-gonadal (HPG) reproductive axis. Kisspeptins are reported to be the most potent activators of HPG axis known to date. Kisspeptin increases intracellular calcium concentration ([Ca2+]i) in immortalized GnRH neurons (1). Whether RFamide peptides modulate the effects of kisspeptins on GnRH neurons has not been investigated. The goal of the present study was to examine the effects of RFamide related peptide-1 (RFRP-1), a mammalian orthologous peptide of GnIH, on kisspeptin-stimulated gonadotropin-releasing hormone (GnRH) neurons. In the study the GT1-7 cell line expressing GnRH was used as model to explore the effects of RFRP-1 on kisspeptin-activated GnRH neurons. The effects of RFRP-1 on GnRH release and calcium signaling in GT1-7 cells were investigated. GT1-7 cells were cultured on poly-D-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 μM Fura-2 AM, [Ca2+]i responses were quantified by the changes in 340/380 nm ratio for individual GT1-7 cells. GnRH release to medium was assayed by ELISA. Kisspeptin-10 at 100 nM (n=65) caused a significant increase (P<0.01) in GnRH release. Co- treatment with kisspeptin in the presence of 1 µM RFRP-1 (n=43) attenuated (P<0.05) kisspeptin-induced GnRH release in GT1-7 cells. Kisspeptin-10 at 100 nM caused a significant increase (p<0.001) in [Ca2+]i compared to basal levels in GT1-7 cells. RFRP-1 did not cause any change in kisspeptin-induced [Ca2+]i increase in GT1-7 cells. The results suggest that RFRP-1 may have inhibitory effects on kisspeptin-activated GnRH neurons without affecting [Ca2+]i.