Kisspeptin is a potent stimulator of GnRH secretion that has been implicated in the feedback actions of ovarian steroids. In ewes, the majority of hypothalamic kisspeptin neurons are found in the arcuate nucleus (ARC), with a smaller population located in the preoptic area. Most arcuate kisspeptin neurons express estrogen receptor-alpha, as do a set of arcuate neurons that contain both dynorphin and neurokinin B (NKB), suggesting that all three neuropeptides are colocalized in the same cells. In this study we tested this hypothesis using dual immunocytochemistry and also determined if kisspeptin neurons contain MSH or agouti-related peptide. To assess colocalization of kisspeptin and dynorphin, we used paraformaldehyde-fixed tissue from estrogen-treated ovariectomized ewes in the breeding season (n = 5). Almost all ARC, but no preoptic area, kisspeptin neurons contained dynorphin. Similarly, almost all ARC dynorphin neurons contained kisspeptin. In experiment 2 we examined colocalization of kisspeptin and NKB in picric-acid fixed tissue collected from ovary intact ewes (n = 9). Over three quarters of ARC kisspeptin neurons also expressed NKB, and a similar percentage of NKB neurons contained kisspeptin. In contrast, no kisspeptin neurons stained for MSH or agouti-related peptide. These data demonstrate that, in the ewe, a high percentage of ARC kisspeptin neurons also produce dynorphin and NKB, and we propose that a single subpopulation of ARC neurons contains all three neuropeptides. Because virtually all of these neurons express estrogen and progesterone re-ceptors, they are likely to relay the feedback effects of these steroids to GnRH neurons to regulate reproductive function.