The neuropeptide kisspeptin plays an important role in fertility and the onset of puberty, stimulating gonadotropin-releasing hormone (GnRH) neurons to activate the hypothalamic–pituitary–gonadal axis. Several studies have demonstrated a morphological interaction between kisspeptin- and GnRH-expressing neurons; however, few have addressed the interaction of kisspeptin with other neuronal subtypes. We recently showed that fibers immunoreactive for kisspeptin were densely distributed in the dorsal part of the arcuate nucleus. These fibers were found in close proximity to GnRH and tuberoinfundibular dopamine (TIDA) neurons. In the present study, using biotinylated kisspeptin, we established a visualization method for identifying kisspeptin binding sites on TIDA neurons. Biotinylated kisspeptin bound to the cell bodies of TIDA neurons and surrounding fibers, suggesting that TIDA neurons express sites of action for kisspeptin. Our assay also detected biotinylation signals from kisspeptin binding to GnRH fibers in the median eminence, but not to cell bodies of GnRH neurons in the medial preoptic area. Positive signals were completely eliminated by addition of excess non-labeled kisspeptin. This method enabled us to detect kisspeptin binding sites on specific neural structures and neuronal fibers.