Kisspeptin is recognized to play a critical role in eliciting the pubertal resurgence of pulsatile GnRH release, the proximal trigger of puberty in higher primates. Expression of the kisspeptin receptor (GPR54) by GnRH neurons indicates a direct action of kisspeptin on the GnRH neuronal network. The purpose of the present study was to examine the distribution of kisspeptin cell bodies in the monkey hypothalamus and to assess the structural basis for the stimulatory action of kisspeptin on the GnRH neuronal network. Three castrated male rhesus monkeys, 39-51 months of age, were deeply anesthetized and their brains perfused transcardially with 4% paraformaldehyde in PBS. Serial 25-microm coronal sections throughout the hypothalamus were prepared, and immunopositive neurons identified using a cocktail of specific primary antibodies (sheep anti-kisspeptin at 1:120,000, and rabbit anti-GnRH at 1:100,000) detected with fluorescently tagged secondary antibodies (antisheep, Alexa Fluor 488; antirabbit, Cy3) in combination with confocal microscopy. Kisspeptin perikarya were found only in the mediobasal hypothalamus (MBH) almost exclusively in the posterior two-thirds of the arcuate nucleus. Surprisingly, kisspeptin-beaded axons made only infrequent contacts with GnRH neurons (kisspeptin and GnRH profiles abutting in a 0.5- to 1.0-mum optical section) in the MBH. In the median eminence, kisspeptin and GnRH axons were found in extensive and intimate association. GnRH contacts on kisspeptin perikarya and dendrites were observed. These findings indicate that nonsynaptic pathways of communication in the median eminence should be considered as a possible mechanism of kisspeptin regulation of GnRH release, and provide an anatomical basis for reciprocal control of kisspeptin neuronal activity by GnRH.