The HRP tracing method was employed to investigate the organization and afferent connections of the interpeduncular nucleus (IPN) in the rat. To study the topographical features of the different projections, a method was devised for obtaining HRP placements of limited size in different areas of the IPN. The main afferent connection of the IPN is a topographically organized projection from the medial habenula (Hb). This projection follows a reversed caudorostral pattern, terminating throughout all but the caudalmost part of the IPN. The dorsal part of the IPN receives a sparse innervation arising mainly from a narrow lateral and ventrolateral area of the medial Hb. The ventral two thirds of the IPN receives a much heavier projection, as follows: A large ventrolateral area of the medial Hb projects to the lateral part of the IPN in a completely bilateral way. An additional projection, which is predominantly ipsilateral, arises from the rostral half of the dorsolateral part of the medial Hb and terminates in the caudal IPN. The medial part of the medial Hb projects preferentially to central areas of the IPN. The projection from the lateral Hb is quantitatively much smaller but appears to be distributed to the entire length of the IPN, following a nonreversed caudorostral arrangement, with the ipsilateral projection predominating. The projections from the medial and lateral Hb to the IPN were confirmed by tracing anterogradely transported HRP as well. No reciprocal connection from the IPN to the Hb could be demonstrated. A sparse projection to the IPN with a strong ipsilateral predominance arises from the horizontal limbs of the nucleus of the diagonal band of Broca. This was the only projection observed from the septal region. Sparse projections from the premammillary and supramammillary nuclei were also demonstrated. Confirmatory data and some details of organization were also obtained for projections to the IPN from other areas, including the medial and dorsal raphe nuclei, the dorsal tegmental nucleus of Gudden, and the adjacent dorsolateral tegmental nucleus. Very small projections from the ventral tegmental nucleus and the locus coeruleus were also found.