In female mammals, lactation suppresses GnRH/LH secretion resulting in transient infertility. In rats, GnRH/LH secretion is recovered in 18-48 hours (h) after pup separation (PS) and rapidly re-suppressed by subsequent re-exposure of pups. In order to elucidate the mechanisms underlying these rapid modulations in GnRH/LH secretion, changes in the expression of kisspeptin, a stimulatory neuropeptide for GnRH secretion, were examined by in situ hybridization in several lactating conditions (normal-lactating; 4h PS; 18h PS; 4h PS + 1h re-exposure of pups; non-lactating). 4h or 18h PS significantly increased kiss1 expressing neurons in both the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), and subsequent 1h exposure of pups after 4h PS re-suppressed kiss1 expression in the AVPV. Change in kiss1 expression was observed prior to the changes in GnRH/LH, indicating that the changes in GnRH/LH secretion result from the change in its upstream modulator, kisspeptin. We further examined the mechanisms underlying the rapid modulation of kiss1 expression. Since the changes in serum prolactin were observed under these lactating conditions in rough inverse relationship with kiss1 expression, we examined the effect of prolactin on kiss1 expression. Continuous intravenous administration of prolactin for 1h significantly suppressed kiss1 expression in the AVPV but not in the ARC. We also examined the possibility that the suckling stimulus modulate the kiss1 expression in the kisspeptin neurons through ascending sensory input. Injection of the anterograde tracer to the subparafascicular parvocellular nucleus (SPFpc) in midbrain which relay suckling stimulus revealed direct neuronal connections between the PSFpc and kisspeptin neurons in both the AVPV and ARC. These results indicate that suckling stimulus rapidly modulate kiss1 expression directly via neuronal connections in both the AVPV and the ARC, and partially through serum prolactin, resulting in modulation in GnRH/LH secretion.