The topographical and ultrastructural organization of the habenular projection to the interpeduncular nucleus (IPN) of the rat was examined employing the anterogradely transported tracer wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and the chromogen tetramethylbenzidine (TMB). Unilateral placements of WGA-HRP in the habenular complex resulted in heavy terminal labelling in the rostral, central, and intermediate subnuclei bilaterally, and in the lateral subnuclei ipsilaterally. The apical subnucleus possessed only a sparse amount of label. Placements confined to the medial habenula (mH) produced similar results to those observed when the entire habenula was filled, suggesting that the afferent contribution made by the lateral habenula (lH) to the IPN is small. Unilateral placements of WGA-HRP in the dorsal portion of the mH resulted in heavy, predominantly ipsilateral labelling in the lateral subnucleus and the dorsal cap of the rostral subnucleus. In the lateral subnucleus labelled habenular terminals consistently contacted single dendritic processes shared by one or more other boutons, possibly of nonhabenular origin. Labelled habenular terminals in the rostral subnucleus normally contacted one or two dendrites. Labelled terminals in both subnuclei possessed clear, spherical vesicles and a variable number of dense-core vesicles. Unilateral placements of WGA-HRP in the ventral portion of the mH resulted in heavy labelling in the rostral half of the rostral subnucleus with a slight ipsilateral predominance, and in the central and intermediate subnuclei bilaterally. Terminal labelling was observed in crest and S synapses in the intermediate and central subnuclei respectively. Crest synapses, which consist of two parallel habenular terminals contacting an attenuated dendritic process, normally possessed label in only one of the two boutons. In the central subnucleus labelled horizontal axons formed several en passant S synapses with dendritic processes of small and medium diameter. These synaptic specializations of habenular axons contained numerous clear, spherical vesicles. This study demonstrates that a major topographically organized projection to the IPN originates from two distinct subpopulations of habenular neurons which comprise a dorsal sector and a ventral sector of the mH. Ultrastructural examination demonstrated that axons originating from neurons in the ventral and dorsal mH form characteristic contacts in the various IPN subnuclei.