Neuropeptide B (NPB) and the structurally related neuropeptide W (NPW) have recently been identified as the endogenous ligands of the orphan G protein-coupled receptors GPR7 and GPR8. Whereas NPW is a high-affinity ligand for both GPR7 and GPR8, NPB activates only GPR7 in sub-nanomolar concentrations. GPR7 is highly conserved in both human and rodent orthologs while GPR8 has not been found in rodents. GPR7 mRNA is expressed in discrete regions of the hypothalamus suggesting a role in the regulation of energy homeostasis and neuroendocrine axes. In the present study, we have generated and extensively characterized antibodies that exert selective specificity for NPB. In dot-blot assays, these antibodies detected NPB but not NPW. Immunofluorescent staining of rat brain sections revealed moderately dense plexus of NPB-immunoreactive fibers and terminals in discrete areas of the hypothalamus. Neuronal somata were only seen in colchicine-treated rats. This immunostaining was completely abolished by preincubation of the antibodies with NPB but not with NPW. NPB-immunoreactivity was enriched in many regions within the hypothalamus which also contained high levels of GPR7 mRNA including the ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, supraoptic retrochiasmatic nucleus, and in the area ventral to the zona incerta. Together, NPB and its receptor GPR7 exist in close proximity in the rat hypothalamus and are, hence, ideally positioned to modulate neuroendocrine functions.