The organization of the brainstem serotonin neuron projection to the hippocampal formation was analyzed in the rat. This projection arises in the raphe nuclei of the midbrain. Following destruction of the midbrain raphe nuclei, chiefly nucleus centralis superior, there is a 72% decrease in hippocampal serotonin content. Injection of tritiated amino acid into the midbrain raphe nuclei results in transport of tritiated protein to the hippocampal formation and this transport is blocked in animals pretreated by intraventricular administration of 5,6-dihydroxytryptamine (5,6-DHT). Autoradiographic analysis indicates that the transport reaches the hippocampal formation primarily via two major pathways, the cingulum and the fornix. Cingulum fibers terminate predominantly in the dorsal hippocampus whereas the fornix distributes throughout the entire hippocampal formation. Some fibers reach the ventral hippocampus from the entorhinal area. Within the hippocampus there is dense labeling in a restricted lamina of the CA1 stratum lacunosum-moleculare with moderate labeling in stratum radiatum. Stratum oriens is sparsely labeled in CA1 and moderately so in CA2 and CA3. Stratum radiatum and stratum lacunosum-moleculare are moderately densely labeled in CA2 and Ca3. The area dentata is sparsely to moderately labeled in the molecular layer and heavily labeled in a thin lamina of the hilar zone immediately beneath the granule cell layer. The remaining hilar zone is moderately labeled. All of the discrete labeling of the hippocampus and area dentata described above is absent in animals pretreated with 5,6-DHT. These observations indicate that serotonin neurons of the midbrain raphe provide a highly organized innervation of the hippocampal formation in the rat.