The spinal cord is innervated by brainstem serotonergic neurons, some of which contain substance P and/or thyrotropin-releasing hormone in addition to serotonin. These neurons project at least three types of axons to the spinal cord: those containing both substance P and thyrotropin-releasing hormone, those containing thyrotropin-releasing hormone but not substance P, and those containing neither substance P nor thyrotropin-releasing hormone. However, the organization of the different types of serotonergic processes is unclear. In the present studies, the types of serotonergic axons projecting to two kinds of spinal neurons were examined. Somatic and parasympathetic preganglionic motoneurons were labeled retrogradely from the pelvic or sciatic nerve, respectively. Sections containing these neurons were stained either for serotonin and substance P, or for serotonin and thyrotropin-releasing hormone. Of a total of 428 profiles examined that were retrogradely labeled from the sciatic nerve, 425 (99%) were apposed by serotonin-immunoreactive varicosities; similarly, of a total of 382 profiles examined that were retrogradely labeled from pelvic nerve, 353 (92%) were apposed by serotonin-immunoreactive varicosities. However, differences appeared to exist between the types of serotonergic varicosities innervating these two groups of neurons. Among the profiles labeled from the sciatic nerve, it was estimated that over 97% were apposed by serotonin-immunoreactive varicosities in which serotonin co-existed with substance P and thyrotropin-releasing hormone. In contrast, among the profiles labeled from pelvic nerve that were apposed by serotonin-immunoreactive varicosities, it was estimated that less than 1% were apposed by serotonin-immunoreactive varicosities containing both thyrotropin-releasing hormone and substance P. We estimate that most of the remainder (about 80%) were apposed by serotonin-immunoreactive varicosities containing thyrotropin-releasing hormone but not substance P. We conclude that both the cell bodies of neurons retrogradely labeled from the pelvic nerve and those labeled from the sciatic nerve were apposed by serotonin varicosities. However, these two systems of neurons appear to be innervated largely by two different populations of serotonergic cells. This suggests that the raphe-spinal serotonergic system may independently modulate the activities of somatic motoneurons and parasympathetic preganglionic motoneurons.