The distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) previously shown to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[125I]VIP specific binding was maximal within 90-120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity (Kd of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides "peptide-histidine-isoleucine" (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide greater than PHI greater than secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.