Melatonin is secreted at night by the pineal gland and governs the reproductive system in seasonal breeders, such as sheep. The mechanism by which melatonin regulates reproduction is not known. The circannual rhythmicity of other factors including prolactin, is also regulated by photoperiod through changes in melatonin secretion. In sheep, plasma prolactin levels are higher in the non-breeding season than the breeding season. Kisspeptin, synthesised by neurons in the ovine arcuate nucleus (ARC) and preoptic area, is a key regulator of reproduction through stimulation of gonadotropin releasing hormone (GnRH) secretion and its expression in the ARC is reduced during the non-breeding season. We hypothesised that kisspeptin expression is directly, or indirectly, regulated by melatonin and/or prolactin. We first examined the expression of melatonin receptor (MTNR1A) in kisspeptin (Kiss1 mRNA) neurons in the ARC of ovariectomised (OVX) sheep using double-label in situ hybridisation. MTNR1A mRNA was not expressed by kisspeptin neurons, while strong expression was detected in the pars tuberalis. We then examined the expression of the long-form prolactin receptor (PRLR-L) in ARC kisspeptin neurons. In OVX ewes, approximately 60 of kisspeptin neurons expressed PRLR-L mRNA at similar levels in the breeding and non-breeding seasons. We then sought to determine whether prolactin treatment during the breeding season regulates kisspeptin expression in the ARC. Continuous central infusion of prolactin (20 mug/h for 7 days) in oestradiol-treated OVX sheep did not alter Kiss1 mRNA expression or LH secretion, but induced substantial pSTAT-ir nuclei staining in the mediobasal hypothalamus. We conclude that the seasonal change in kisspeptin neurons cannot be regulated directly by melatonin, but may be due to changes in prolactin levels. Despite this, kisspeptin expression was unchanged after exogenous prolactin treatment in breeding season ewes.