We examined the role of kisspeptin and its receptor, the G-protein-coupled receptor GPR54, in governing the onset of puberty in the mouse. In the adult male and female mouse, kisspeptin (10–100 nm) evoked a remarkably potent, long-lasting depolarization of >90% of gonadotropin-releasing hormone (GnRH)–green fluorescent protein neurons in situ. In contrast, in juvenile [postnatal day 8 (P8) to P19] and prepubertal (P26–P33) male mice, kisspeptin activated only 27 and 44% of GnRH neurons, respectively. This developmental recruitment of GnRH neurons into a kisspeptin-responsive pool was paralleled by an increase in the ability of centrally administered kisspeptin to evoke luteinizing hormone secretion in vivo. To learn more about the mechanisms through which kisspeptin–GPR54 signaling at the GnRH neuron may change over postnatal development, we performed quantitative in situ hybridization for kisspeptin and GPR54 transcripts. Approximately 90% of GnRH neurons were found to express GPR54 mRNA in both juvenile and adult mice, without a detectable difference in the mRNA content between the age groups. In contrast, the expression of KiSS-1 mRNA increased dramatically across the transition from juvenile to adult life in the anteroventral periventricular nucleus (AVPV; p < 0.001). These results demonstrate that kisspeptin exerts a potent depolarizing effect on the excitability of almost all adult GnRH neurons and that the responsiveness of GnRH neurons to kisspeptin increases over postnatal development. Together, these observations suggest that activation of GnRH neurons by kisspeptin at puberty reflects a dual process involving an increase in kisspeptin input from the AVPV and a post-transcriptional change in GPR54 signaling within the GnRH neuron.