It has previously been shown that the median raphe nucleus (MR) is a source of pronounced projections to the septum and hippocampus. The present study examined collateral projections from MR to the medial septum (MS) and to various regions of the hippocampus. The fluorescent retrograde tracers, Fluororuby and Fluorogold, were injected into the septum and hippocampus, respectively, and the median raphe nucleus was examined for the presence of single- and double-labeled neurons. The dorsal raphe nucleus (DR) was also examined for the presence of single- and double-labeled cells and comparisons were made with the MR. The main findings were: (1) pronounced numbers of retrogradely labeled cells (approximately 50 cells/section) were present in MR with injections in the MS or in various regions of the hippocampus; (2) approximately 8-12% of MR cells were double-labeled following paired injections in the MS-CA1, MS-CA3, and MS-dentate gyrus of the dorsal hippocampus, the lateral MS-dentate gyrus, and the MS-ventral hippocampus; (3) single- and double-labeled cells were intermingled throughout MR and present in greater numbers in the rostral than caudal MR; and (4) significantly more single- and double-labeled cells were present in MR than in DR with all combinations of injections. These findings demonstrate that MR projects strongly to the MS and hippocampus, and that a significant population of MR neurons (8-12%) sends collateral projections to both sites. It is well established that the MR nucleus serves a direct role in the desynchronization of the electroencephalographic (EEG) activity of the hippocampus-or the blockade of the hippocampal theta rhythm. The MR neurons that we have identified with collateral projections to the septum and hippocampus may be critically involved in the modulation/control of the hippocampal EEG. A role for the MR in memory associated functions of the hippocampus is discussed.