High levels of circulating prolactin are known to cause infertility, but the precise mechanisms by which prolactin influences the neuroendocrine axis are yet to be determined. We used dual-label in situ hybridization to investigate whether prolactin-receptor (PRLR) mRNA is expressed in GnRH neurons. In addition, because γ-aminobutyric acidergic and kisspeptin neurons in the rostral hypothalamus are known to regulate GnRH neurons and, hence, might mediate the actions of prolactin, we investigated whether these neurons coexpress PRLR mRNA. (35)S-labeled RNA probes to detect PRLR mRNA were hybridized together with digoxigenin-labeled probes to detect either GnRH, Gad1/Gad2, or Kiss1 mRNA in the rostral hypothalamus of ovariectomized (OVX), estradiol-treated rats. Additional sets of serial sections were cut through the arcuate nucleus of OVX rats, without estradiol replacement, to examine coexpression of PRLR mRNA in the arcuate population of kisspeptin neurons. PRLR mRNA was highly expressed throughout the rostral preoptic area, particularly in periventricular regions surrounding the third ventricle, and there was a high degree of colocalization of PRLR mRNA in both Gad1/Gad2 and Kiss1 mRNA-containing cells (86 and 85.5%, respectively). In contrast, only a small number of GnRH neurons (<5%) was found to coexpress PRLR mRNA. In the arcuate nucleus of OVX rats, the majority of Kiss1 mRNA-containing cells also coexpressed PRLR mRNA. These data are consistent with the hypothesis that, in addition to a direct action on a small subpopulation of GnRH neurons, prolactin actions on GnRH neurons are predominantly mediated indirectly, through known afferent pathways.