RFamide related peptides (RFRP)-1 and RFRP-3 are neuropeptides derived from the same preproprotein. We have examined the distribution of RFRP-1 and RFRP-3 immunoreactivities (irs) in the rat central nervous system using specific antibodies. Neuronal cell bodies containing both RFRP-1 and RFRP-3 were detected within the caudal portion of the hypothalamus, the periventricular nucleus (PerVN), and the portion around or above the ventromedial nucleus of the hypothalamus. Both immunohistochemical and in situ hybridization analyses showed that neurons containing RFRP immunoreactivity and mRNA were distinct from those of neuropeptide FF, which contains the same structure at the C-terminus, Pro-Glu-Arg-Phe-NH2, as RFRP-3. Fibers containing both RFRP-1 and RFRP-3 were widely distributed in the brain: the lateral septal nucleus in the telencephalon, the paraventricular thalamic nucleus, various hypothalamic nuclei, the periaqueductal gray in the midbrain, the parabrachial nucleus in the pons, and the nucleus tractus solitarius (NTS) in the medulla oblongata. Only RFRP-1-ir was detected within the posterior gray horn in the spinal cord. Only RFRP-3-ir was detected in several thalamic nuclei and the spinal cord, especially at the posterior intermediate sulcus and within the anterior gray horn. Intracerebroventricular administration of RFRPs induced c-Fos expression in the anterior portion of the NTS, locus coeruleus, the nucleus of incertus, supraoptic nucleus, PerVN and the arcuate nucleus of the hypothalamus. These results show that RFRP-1 and RFRP-3 are widely distributed in the rat central nervous system and might be involved in various functions such as the neuroendocrine system or pain modulation.