We previously reported the existence of motilin receptors in the cerebellum of the rabbit. We now explored the existence of motilin receptors in other brain regions and determined their association with neurons by subcellular fractionation studies. Autoradiographic studies with [(125)I]nle13-porcine motilin on rabbit coronal brain sections revealed specific binding sites in the hippocampus, thalamus, hypothalamus and amygdaloid body. Receptor binding studies allowed the identification of two binding sites. In all regions the density of the high-affinity binding site was lower than in the cerebellum, but its affinity was the same, except for the hypothalamus. No differences were found for affinity or density of the low-affinity receptor site. Homogenates of rabbit cerebellum were subjected to differential centrifugation. The highest motilin binding (10-times more than in the postnuclear supernatant) was found in the fraction which also showed maximal enrichment of [11-3H]saxitoxin binding (selective marker for voltage-sensitive Na+ channels), 6.9-fold, and cytochrome c oxidase activity (mitochondrial marker), 2.4-fold. In discontinuous sucrose density gradient centrifugation the motilin and saxitoxin binding both peaked in the 0.85-1 M layer, while cytochrome c oxidase was maximal in the 1.2 M layer. In conclusion, motilin receptors exist in several regions of the rabbit brain and are probably associated with synaptosomes. These findings further support a neurotransmitter role for motilin in the brain.