The projection pathways of neurons containing melanin-concentrating hormone (MCH) and neuropeptide EI (NEI), two peptides colocalized in the lateral hypothalamic area (LHA) of the rat, were mapped using the retrogradely transported fluorescent dyes, true blue (TB) and diamidino yellow (DY). TB and DY were injected into the medial septum/diagonal band complex (MS/DBC) and the thoracic level of the spinal cord (SpCd), respectively. Brains from rats receiving only one or both tracer injections were immunohistochemically stained for MCH in the spinal cord and NEI in the forebrain. In the MS/DBC, NEI-immunoreactive (-ir) fibers are concentrated in the MS and in the vertical and horizontal limbs of the DBC. In the SpCd, MCH-ir fibers are concentrated primarily in lamina X. Of the diencephalic NEI-ir neurons, 37.15% project to the MS/DBC and reside in the rostromedial zona incerta (ZIm), in the LHA_t and LHA_p, and in the perifornical region. Of the diencephalic MCH-ir neurons, 20.2% project to the SpCd and reside in the LHA_t and LHA_p. In addition, 2.2% of the MCH-ir cells and 8.7% of the NEI-ir cells in the hypothalamus were labeled with both retrograde tracers and thus project to both the MS/DBC and SpCd. These dual projection neurons are located mainly in the LHA_t and LHA_p. Anterograde injections of the tracer Phaseolus vulgaris leucoagglutinin into the LHA_t and ZIm corroborated our findings in the retrograde studies. Potential autonomic and behavioral roles of the NEI and MCH systems in the MS/DBC and the SpCd are discussed.