Fertility of high producing dairy cattle is very low, most likely related to the extreme metabolic demands associated with high milk production. To address this issue, a better understanding of the brain control of reproduction in dairy cattle is needed. In cattle, Kp treatment stimulates gonadotrophin secretion, while RFRP-3 inhibits it. Conversely, RFRP-3 mRNA levels are stimulated by reduced feed intake. We have used immunohistochemistry to map the distribution of Kp and RFRP-3 neurons in the hypothalamus of dairy cattle, determined their co-expression with ERα, and whether they make apparent contact with GnRH neurons. The heads of male and female calves (n=3 each), steers, heifers and lactating cows (n=2/group) were perfusion fixed (4% paraformaldehyde) and the brains dissected out and frozen. Dual label immunohistochemistry for Kp or RFRP-3 with ERα or GnRH was performed on 40µm cryostat sections, using fluorescently labelled secondary antibodies. Kp neurons were located primarily in the arcuate nucleus, with some cells in the preoptic area, although few were observed in the calves. Most Kp neurons in the adult animals of both sexes, but few in the calves, expressed ERα. We were unable to determine whether Kp neurons contact GnRH neurons. RFRP-3 neurons were localised in the dorsomedial hypothalamus and paraventricular nucleus, with fewer cells observed in the cows than the heifers & steers. ERα was not expressed in this region. Fewer than 20% of GnRH neurons received close contact from RFRP-3 neurons, with a few RFRP-3 neurons receiving apparent contact from GnRH fibres. These data show that Kp and RFRP-3 neurons in dairy cattle are neuroanatomically like other known species, but further studies are required to clarify their physiological role.