The organization of the interpeduncular nucleus (IPN) in the adult rat was analyzed using cytoarchitectonic, histochemical and immunohistochemical methods. Four paired and four unpaired subnuclei can be distinguished in the IPN on the basis of neuronal size, morphology, staining characteristics and packing density. The rostral portion of the IPN contains a rostral dorsal, a rostral ventral and paired rostral lateral and dorsal lateral nuclei. The dorsal lateral nuclei continue into the caudal IPN, which also contains a caudal dorsal, a caudal ventral and paired caudal lateral nuclei. The distribution of extrinsic afferents and of chemically identified intrinsic neuronal and fiber populations within subdivisions of the IPN was examined using immunohistochemistry, acetylcholinesterase histochemistry, catecholamine histofluorescence and the autoradiographic tracing method. Six immunohistochemically distinct neuronal groups are identified in the IPN. Perikarya and axons showing substance P-, leu-enkephalin-, somatostatin-, avian pancreatic polypeptide-, serotonin- and glutamic acid decarboxylase-like immunoreactivity are localized to specific IPN subnuclei. Acetylcholinesterase-positive staining, extrinsic norepinephrine-containing fibers and afferents from the dorsal tegmental nuclei are also distributed specifically to IPN subnuclei. These findings demonstrate a cytoarchitectonic and cytochemical complexity in the rat IPN that implies an important functional role for this poorly understood nuclear complex.