The anatomical localization of neuropeptide FF receptors was examined by in vitro autoradiography techniques in rat brain sections by using [125I][D.Tyr1, (NMe)Phe3]NPFF. The specific binding of [125I][D.Tyr1, (NMe)Phe3]NPFF reached 90% of the total binding at 0.05 nM in rat spinal cord sections. Up to 40% of the specific binding of[125I][D.Tyr1, (NMe)Phe3]NPFF to rat spinal cord sections was still detectable following fixation with glutaraldehyde. Afterwards, the distribution of NPFF receptors was studied by light microscopy and their densities by microdensitometry with an image analysis system. In the light microscope, [125I][D.Tyr1, (NMe)Phe3]NPFF labelling appeared more or less uniformly distributed over nerve-cell bodies and surrounding neuropil. High concentrations of binding sites were detected in the presubiculum, parafascicular thalamic nucleus, gracile nucleus, spinal trigeminal tract nucleus, and a number of brainstem nuclei, with virtually no labelling in the cerebellum. In several areas a rostrocaudal gradient of sites concentration was observed. Neuropeptide FF receptors are well-placed to control incoming sensory and autonomic information processing. In contrast, the more recently developed areas of the forebrain possessed low density of sites. The distribution of [125I][D.Tyr1, (NMe)Phe3]NPFF binding sites should suggest anatomical substrates for the actions of neuropeptide FF.