This study investigated the synaptic interactions between hypoglossal motoneurons that project to the genioglossus muscle and substance P (SP) containing immunoreactive nerve terminals. Cholera toxin B conjugated to horseradish peroxidase (CTB-HRP) was injected into the right half of the genioglossus muscle in four anesthetized cats. Two days later, the animals were perfused with acrolein fixative. Tetramethylbenzidine (TMB) was the chromogen used to detect retrogradely labeled cells containing CTB-HRP. The tissues were then processed for immunocytochemistry using an antiserum raised against SP with diaminobenzidine (DAB) as the chromogen. At the light microscopic level, labeled cells were observed primarily ipsilaterally in ventral and ventrolateral subdivisions of the hypoglossal nucleus. The majority of these labeled cells were observed at the level of the area postrema. At the electron microscopic level, SP-like immunoreactive nerve terminals formed synaptic contacts with retrogradely labeled dendrites and perikarya. Nineteen percent of the terminals that contacted retrogradely labeled cells contained SP. These are the first ultrastructural studies demonstrating synaptic interactions between protruder hypoglossal motoneurons and SP terminals. These studies demonstrate that hypoglossal motoneurons which innervate the major protruder muscle of the tongue, the genioglossus muscle, may be modulated by SP. Thus, SP may play a role in the control of protrusive movements of the tongue acting via neurokinin receptors.