The habenular complex of the epithalamus connects the limbic basal forebrain with numerous neuromodulatory centers in the midbrain. The habenula consists of the medial and lateral nuclei, each of which is speculated to contain multiple subdivisions. Such anatomical arrangements raise the possibility that the habenula accounts for multiple channels of information flow from the limbic forebrain to the midbrain. For understanding whether and, if so, how the multiple streams are organized via the habenula, knowledge of the precise input–output connectivity of each habenular subdivision is essential. In the present study, biotinylated dextran amine and cholera toxin subunit B were used to delineate the differential outputs of various subregions of the medial and lateral nuclei of the habenula in the rat. Both anterograde and retrograde tracing uncovered a heavy commissural connection between the two habenulae on the ipsilateral and contralateral sides. The commissural projection arises primarily in the lateral nucleus and exhibits a fine topography in that a local commissural efferent terminates primarily in the corresponding subregion on the contralateral side. The subregions of the medial and lateral nuclei also give rise to distinct projections to midbrain areas such as the interpeduncular nucleus, the median/paramedian nuclei, and the central gray. These projections produce terminal fields centered in different areas of the targets, supporting the topographically organized descending projections from the habenula. These data together support the organization of multiple channels in the habenula that convey parallel streams of information to the contralateral habenula, midbrain, and brainstem. J. Comp. Neurol. 513:173–187, 2009. © 2009 Wiley-Liss, Inc.