A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.