Kisspeptin is a pivotal regulator of the onset of puberty and the estrus cycle, but may also take part in pregnancy and lactation. Kisspeptin neurons and their fibers are distributed abundantly throughout the arcuate nucleus (ARC) of the hypothalamus, but the targets of the fiber projections in the ARC have not been fully investigated. The present study followed the projection of kisspeptin fibers to tuberoinfundibular dopaminergic (TIDA) neurons in the ARC, pivotal endocrine neurons that control prolactin secretion. Immunoreactive fibers of kisspeptin or neurokinin B, a peptide coexpressed in kisspeptin neurons, were abundantly found adjacent to TIDA neurons in female rats, but few were observed in male rats. The immunoreactivities of both peptides adjacent to TIDA neurons were significantly reduced in estradiol-primed ovariectomized rats. Precise 3D analysis of the attachment of kisspeptin-immunoreactive fibers to TIDA neurons was achieved using a synaptic marker that indicated synaptic connection. Finally, double-labeling immunoelectron microscopy confirmed the synaptic connections of kisspeptin-immunoreactive fibers to the cell body and fibers of TIDA neurons. These findings indicate that in female rats, kisspeptin/NKB fibers may directly affect TIDA neurons that regulate prolactin secretion, and that they are more likely to be activated during low estradiol status. (c) 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.