Central administration of neurokinin B (NKB) agonists stimulates immediate early gene expression in the hypothalamus and increases secretion of vasopressin from the posterior pituitary through a mechanism that depends on the activation of neurokinin receptor 3 receptors (NK3R). Here we report that, in the rat, immunoreactivity for NK3R is expressed in magnocellular vasopressin and oxytocin neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus, and that NKB immunoreactivity is expressed in fibres in close juxtaposition with vasopressin neurones at both of these sites. Retrograde tracing in the rat showed that some NKB-expressing neurones in the arcuate nucleus project to the SON, and in mice, using an anterograde tracing approach, we found that kisspeptin-expressing neurones of the arcuate nucleus, which are known to co-express NKB, project to the SON and PVN. Finally, we show that i.c.v. injection of the NK3R agonist senktide potently increases the electrical activity of vasopressin neurones in the SON in vivo with no significant effect detected on oxytocin neurons. The results suggest that NKB-containing neurones in the arcuate nucleus regulate the secretion of vasopressin from magnocellular neurones in rodents, and we discuss the possible significance of this. This article is protected by copyright. All rights reserved.