Pulsatile secretion of hypothalamic gonadotropin-releasing hormone (GnRH) is pivotal for pituitary gonadotrope function. Although the physiological importance of pulsatile GnRH secretion has been well recognized, the mechanisms underlying GnRH pulse generation still remain elusive. We demonstrate rhythmic GnRH gene transcription in single GnRH neurons in coronal slices of the preoptic area (POA) prepared from transgenic mice (postnatal 5-7 days) expressing a GnRH promoter-driven destabilized luciferase reporter (GnRHp-dsLuc). GnRH promoter activity was monitored by real-time bioluminescence device. GnRH secretion was simultaneously measured from the same tissue explant. Although GnRH promoter activity in each GnRH neuron exhibited irregular and episodic, but distinct ultradian oscillations with an interpulse interval of ∼10 hr, GnRH neuronal population showed partially synchronized bursts of GnRH transcriptional activity with ∼2 hr intervals under the basal condition. Intermittent administration of kisspeptin (10nM, 15 min-on, 45 min-off), a potent GnRH secretagogue, evoked dramatic synchronous activation of GnRH gene transcription together with robust stimulation of pulsatile GnRH secretion. Kisspeptin-evoked GnRH transcription was attenuated in the presence of 15a (GPR54 antagonist) or Go6983 (protein kinase C inhibitor). In summary, synchronous burst of kisspeptin-evoked GnRH transcription in hypothalamic neuronal networks appear to be important for GnRH pulse generation.