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The selectivity of two new radioligands, [125I]YVP ([125I]YVPNLPQRF-NH2) and [125I]EYF ([125I]EYWSLAAPQRF-NH2), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF1 and CHO cells expressing hNPFF2 receptors. Saturation binding and displacement experiments showed that [125I]YVP and [125I]EYF bound selectively with a very high affinity, KD=0.18 nM and 0.06 nM, to NPFF1 and NPFF2 receptors respectively.By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF2 binding sites, NPFF1 and NPFF2 receptor distribution was analyzed throughout the rat CNS.The highest densities of [125I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [125I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF1 receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord.The restricted distribution of NPFF1 receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF1 system is absent, there is no strict separation between NPFF1 and NPFF2 system at the supraspinal level. [Copyright &y& Elsevier]