The organization of afferent and efferent connections of the interpeduncular nucleus (IP) has been examined in correlation with its subnuclear parcellation by using anterograde and retrograde tracing techniques. Based on Nissl, myelin, and acetylcholinesterase staining five paired and three unpaired IP subnuclei are distinguished. The unpaired division includes the rostral subnucleus (IP-R), the apical subnucleus (IP-A), and the central subnucleus (IP-C). The subnuclei represented bilaterally are the paramedian dorsal medial (IP-DM) and intermediate subnuclei (IP-I) and the laterally placed rostral lateral (IP-RL), dorsal lateral (IP-DL), and lateral subnuclei (IP-L). Immunohistochemical techniques showed cell bodies and fibers and terminals immunoreactive for substance P, leu-enkephalin, met-enkephalin, or serotonin to be differentially distributed over the different IP subnuclei. Substance P-positive perikarya were found in IP-R, enkephalin neurons in IP-R, IP-A, and the caudodorsal part of IP-C, and serotonin-containing cell bodies in IP-A and the caudal part of IP-L. Efferent IP projections were studied both by injecting tritiated leucine in IP and by injecting HRP or WGA-HRP in the presumed termination areas. The results indicate that the major outflow of IP is directed caudal-ward to the median and dorsal raphe nuclei and the caudal part of the central gray substance, i.e., the dorsal tegmental region. The projection appears to terminate mainly in the raphe nuclei, around the ventral and dorsal tegmental nuclei of Gudden, and in the dorsolateral tegmental nucleus. The descending projection to the dorsal tegmental region originates in virtually all IP subnuclei, but the main contribution comes from IP-R and the lateral subnuclei IP-RL, IP-DL, and IP-L. Sparser projections to the dorsal tegmental region originate in IP-C and IP-I, whereas the contribution of IP-A is only minimal. The projections from IP-R are mainly ipsilateral and those from IP-DM are mainly contralateral. IP fibers to the median and dorsal raphe nuclei originate predominantly in IP-R and IP-DM, and to a lesser extent in IP-C, IP-I, IP-RL, and IP-DL. A much smaller contingent of IP fibers ascends to diencephalic and telencephalic regions. A relatively minor projection, stemming from IP-RL and IP-DL, reaches the lateral part of the mediodorsal nucleus, the nucleus gelatinosus, and some midline thalamic nuclei. These IP fibers follow either the habenulo-interpeduncular pathway or the mammillothalamic tract.(ABSTRACT TRUNCATED AT 400 WORDS)