The molecular mechanisms underlying the reawakening of hypothalamic GnRH neurons at puberty remain to be elucidated. Recently, the G protein-coupled receptor 54 (GPR54) and its endogenous ligand kisspeptin, encoded by the KISS1 gene, have been involved. In fact, GPR54 mutations cause idiopathic hypogonadotropic hypogonadism in human and mice. We used the previously characterized primary culture of human fetal olfactory GnRH-secreting neurons, FNC-B4, to study in vitro the KISS-1/GPR54 regulation. Kisspeptin and GPR54 were immunolocalized in fetal olfactory mucosa, and in FNC-B4. Using confocal microscopy, co-expression of GnRH and GPR54 or GnRH and kisspeptin was found in fetal olfactory mucosa and FNC-B4. The 24 h exposure to sex steroids regulated both gene (qRT-PCR) and protein (western blot and immunocitochemistry) expression of KISS1/GPR54 in FNC-B4. Increasing doses of 17beta-estradiol (0.011 nM) significantly and dose-dependently decreased KISS1/GPR54 mRNA. Conversely, androgens (DHT, 0.011 nM) significantly stimulated KISS1/GPR54 mRNA. Immunofluorescence with anti-kisspeptin confirmed that 1 nM 17beta-estradiol significantly reduced, whereas 1 nM DHT significantly increased, the % of kisspeptin-positive FNC-B4 cells. Testosterone treatment showed no effect, but, blocking its aromatization with letrozole, it mimicked DHT stimulatory activity. In addition, 24 h exposure to leptin (1 nM), an adypocyte-derived hormone acting on the hypothalamus to influence puberty, significantly increased KISS1/GPR54 gene and protein expression. Leptin treatment in FNC-B4 significantly increased also the androgen receptor (AR) mRNA, as well as the mRNA of its own receptor (LEPR), which resulted induced also by 1nM DHT. These data suggest a synergistic action between AR and LEPR to finally up-regulate KISS1/GPR54 system, which, in contrast, was inhibited by estrogen. In conclusion, our results revealed for the first time that sex steroids and leptin regulate KISS-1/GPR54 system in human GnRH neurons, providing new insights into the comprehension of those permissive signals for pulsatile GnRH secretion and puberty onset.