We examined the afferent projections to the subnuclei of the interpeduncular nucleus (IPN) in the rat by means of retrograde and anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). We observed locations of retrogradely labeled cells following injections of WGA-HRP into the IPN, and distributions of anterogradely labeled fibers and terminals within the IPN following injections into the areas that contain cells of origin of afferents. Results of the retrograde and anterograde experiments have clarified the detailed organization of the IPN afferents. A part of the nucleus incertus, located dorsomedial to the dorsal tegmental nucleus, projects to the contralateral half of the rostral subnucleus of the IPN; the pars caudalis of the dorsal tegmental nucleus projects sparsely to the rostral lateral, dorsal lateral, lateral, caudal, and apical subnuclei predominantly contralaterally; the laterodorsal tegmental nucleus, to most of the subnuclei predominantly contralaterally; the ventromedial central gray rostral to the dorsal tegmental nucleus and lateral to the dorsal raphe nucleus projects to the rostral lateral and dorsal lateral subnuclei predominantly contralaterally; the median raphe nucleus, substantially to all subnuclei; the medial habenular nucleus, in a topographic manner, to the rostral, central, and intermediate subnuclei, to the rostral lateral and lateral subnuclei predominantly ipsilaterally, and to the dorsal lateral subnucleus predominantly contralaterally; the supramammillary nucleus and areas around the origin of the mammillothalamic tract and near the third ventricle project sparsely to the ventral part of the rostral subnucleus and to the central, lateral, caudal and apical subnuclei; the nucleus of the diagonal band, sparsely to the rostral, central, dorsal lateral, caudal, and apical subnuclei. These differential projections of the afferents to the subnuclei of the IPN may reflect its complex functions within the limbic midbrain circuit.