Opiate receptors have been localized autoradiographically to many regions of the rat central nervous system. The interpeduncular nucleus has an especially high concentration of these receptors. We used [ 3H]naloxone and [ 125I] [ d-Ala 2, MePhe 4, Glyol 5]enkephalm as ligands to map the distribution of opiate receptors among the subnuclei of the interpeduncular nucleus. The rostral subnucleus contains label that is densest dorsally. More caudally, high densities of opiate binding sites are found in the lateral, rostral, and central subnuclei. The dorsal subnucleus contains a moderate density of binding sites and the intermediate subnuclei contain almost none. Opiate receptors have also been localized to the medial habenulae and the fasciculi retroflexi, which provide a major afferent input to the interpeduncular nucleus. Lesions of the fasciculi retroflexi decreased the density of opiate binding sites in the rostral and lateral subnuclei of the interpeduncular nucleus. There were no changes seen in the dorsal, intermediate or central subnuclei. These results suggest that a minority of opiate receptors in the interpeduncular nucleus are located presynaptically on fasciculi retroflexi axons. Immunocytochemical studies have demonstrated that the rat interpeduncular nucleus contains substance P, serotonin and enkephalin, and each has a distinct subnuclear distribution. Although the opiate binding sites have a wider distribution than substance P, serotonin, or enkephalin individually, the pattern of opiate binding most closely parallels substance P distribution. The combined distribution of substance P, serotonin, and enkephalin is equivalent to that of the opiate binding sites.