Hypothalamic peptide kisspeptin (kp) plays a major role in the initiation of pubertal events in mammals through the activation of gonadotrophin-releasing hormone (GnRH) neurons. The present study was designed to analyse the morpho-functional role of kisspeptin cells in the multimodal effects on GnRH secretion in female prepubertal lambs. The localization of kp immunoreactive (ir) expression was compared with that of GnRH release and neuropeptide Y (NPY) synthesis. The experiment was conducted on eight 32-week-old Merino lambs. The distribution of the peptides was estimated using immunohistochemistry and anti-kp, anti-GnRH and anti-NPY specific antibodies. The hypothalamic area containing numerous kp-ir perikarya and a dense network of kp-ir fibres was localized in the caudal part of the arcuate nucleus. Single axons and single perikarya were observed throughout the hypothalamus extending from the preoptic area to the mammillary bodies. Distinct bundles of kp-ir nerve terminals were observed in the lateral zone of the median eminence (ME) and dispersed in smaller quantities in its medial zone. Double immunostaining indicated that a third of the kp-ir neurons were in close apposition with NPY-ir afferents, and that the distribution of kp-ir nerve terminals in the ME matched that of GnRH-ir nerve terminals. To conclude, the presence of very large numbers of kp-ir neurons in the peripubertal period could be attributed to their stimulating action on the GnRH neuronal system during this period. This influence could act directly in the ME, where both peptides are released. In addition, kp-ir neurons could mediate the effect of NPY on GnRH secretion.