Serotonergic neurons of the medulla oblongata have been proposed to play a role in the control of sensory, motor and autonomic cells in the spinal cord. Many of these raphe neurons have been shown to contain the undecapeptide substance P as well as the tripeptide thyrotropin-releasing hormone, but evidence for the presence of an excitatory arnino acid in these pathways has not yet been documented. In colchicine-treated rats, we have used a combination of retrograde tracing and tri-color immunohistofluorescence techniques to study co-localization of serotonin- and substance P- with glutamate- or aspartate-like immunoreactivities in medullary neurons and the possible spinal projections of these cells. In addition, the distributions of serotonin-, substance P- and glutamate-immunoreactive terminal fields in the dorsal, ventral and lateral horns of the spinal cord were examined with tri-color immunofluorescence in the rat and the primate Macaco fasciculata. In colchicine-treated rats, glutamate- and aspartate-like immunoreactivity was found in practically all serotonin- and substance P-immunoreactive neurons of the B1, B2 and B3 cell groups. Some of these neurons also contained wheat-germ agglutinin conjugated to inactivated horseradish peroxidase and colloidal gold particles retrogradely transported from the spinal cord. In the spinal cords of non-colchicine-treated monkeys and rats, striking co-localization of serotonin, substance P- and glutamate-like immunoreactivities was seen in large boutons, surrounding the dendrites and cell bodies of large alpha motor neurons in the ventral horn. These observations suggest the existence of spinally projecting serotonin/substance P neurons containing excitatory amino acids such as glutamate or aspartate. Thus, these bulbospinal neurons may utilize three types of messenger molecules, an excitatory amino acid, a biogenic amine and one or more peptides. © 1992.