To test the hypothesis that neurons in the hypothalamic paraventricular nucleus (PVN) may be under both direct and indirect regulation by alpha melanocyte-stimulating hormone (alpha-MSH)-synthesizing neurons of the arcuate nucleus, we determined whether the retrogradely transported marker substance, cholera toxin beta-subunit (CtB), when injected into the PVN, labels distinct populations of neurons in the hypothalamic dorsomedial nucleus (DMN) that are innervated by axon terminals containing alpha-MSH. Following iontophoresis of CtB into the PVN, retrogradely labeled neurons were identified in the DMN primarily on the same side as the injection, although a few neurons were also identified in the opposite side of the DMN. The greatest percentage of retrogradely labeled DMN neurons were located in the medial portion of the ventral subdivision of the DMN (DMNv), accounting for approximately 64.8 +/- 1.1% of all CtB-labeled cells in the DMN. The second largest population, comprising 25.9 +/- 1.6% of the total number of CtB cells in the DMN, was diffusely distributed in the dorsal subdivision of the DMN (DMNd). Only 9.4 +/- 0.3% of the CtB-labeled cells were located in the compact zone of the DMN (DMNc). In double-labeling immunofluorescent preparations, 61.1 +/- 1.0% of the CtB cells in the DMNv, 38.6 +/- 0.9% of the CtB cells in the DMNd, and 13.1 +/- 1.3% of the CtB cells in the DMNc were contacted by axon terminals containing alpha-MSH. These data establish that neurons in discrete regions in the DMN may be influenced by the melanocortin signaling system and thereby, could serve as important relay sites to the PVN.