Two-color fluorescence immunohistochemistry was used to identify and map the distribution of nerve processes immunoreactive for both serotonin and substance P in the rat brainstem. Doubly labeled fibers were observed throughout the brainstem, but tended to be densest in cranial nerve motor nuclei and in reticular regions of the ventral medulla. In the trigeminal motor nucleus, the facial nucleus and the spinal accessory nucleus, the majority of serotonergic varicosities also appeared to contain substance P; in the occulomotor nucleus and the hypoglossal nucleus the numbers of double-labeled and single-labeled serotonergic varicosities were roughly equal. Thus, co-existence of substance P with serotonin was common in many cell groups innervating skeletal muscle. The proportion of double-labeled varicosities was significantly lower in the nucleus of the solitary tract, wherein single-labeled varicosities were much more common. Double-labeled fibers and varicosities were also significantly less common in the spinal trigeminal nucleus. In addition, double-labeling appeared to be uncommon in regions involved in the processing of special sensory information (e.g. auditory, vestibular and visual pathways). These results demonstrate a subpopulation of serotonergic fibers immunoreactive for substance P in the brainstem of the rat. The consistently high density of double-labeled processes in cranial nerve motor nuclei suggests that, as may be the case in the spinal cord, neurons containing serotonin and substance P regulate the activity of motoneurons that innervate skeletal muscle. In addition, they may be involved in other aspects of the function of the brainstem.