It is well established that kisspeptin neurons play a critical role in the regulation of the reproductive axis through modulation of the gonadotropin-releasing hormone (GnRH) neurons. There are two functionally distinct populations of kisspeptin neurons in the mouse including the rostral periventricular area of the third ventricle (RP3V) and the arcuate nucleus (ARN). RP3V kisspeptin neurons have been implicated in the generation of preovulatory GnRH surges and the ARN population is proposed to be involved in estrogen negative feedback mechanisms. While there is strong evidence for direct innervation of GnRH neuron somata by RP3V kisspeptin neurons, innervation by ARN kisspeptin neurons is less clear. This study aimed to determine whether ARN kisspeptin neurons project directly to rPOA GnRH neuron somata and proximal dendrites. This was accomplished using Cre-dependent viral tract-tracing specifically out of ARN kisspeptin neurons. Adult Kiss-Cre mice (n=4) were stereotaxically injected with 300nl (5.1x1011 pfu/ml) of Cre-dependent recombinant adenovirus containing farnesylated enhanced green fluorescent (EGFPf) protein into the ARN. Surprisingly, in fixed brain tissue labelled with GFP and GnRH, despite filling reasonable number of kisspeptin neurons with EGFPf in the rostral and caudal regions of ARN (24.5±7.1 and 20.5±9.0/ section respectively), EGFPf-positive fibres were sparse or absent in the rPOA. Moreover, none of the rPOA GnRH neuron somata (38.5±2.1/ section) investigated were apposed by EGFPf-positive fibres. In contrast, extensive EGFPf-positive fibres were observed throughout the ARN coming from ARN kisspeptin neurons and confocal imaging demonstrated close apposition of the fibres and GnRH neuron nerve terminals in the internal zone of median eminence. Our findings clearly show ARN kisspeptin neurons do not project to GnRH neuron somata and proximal dendrites in the rPOA, suggesting ARN kisspeptin neuron regulation of GnRH neurons occurs at more distal portions of the GnRH neuron near the their site of release.