The origin of afferent connections of the hypoglossal nucleus in rats was investigated using horseradish peroxidase (HRP) as a retrograde tracer. Pressure injections (0.15-0.17 mu1) of 15% HRP were introduced into the rostral, middle and caudal portions of the nucleus. Projections to the hypoglossal nucleus originated from 3 regions of the brainstem: the reticular formation, the spinal V complex and the nucleus of the solitary tract. Bilateral projections with ipsilateral predominance came from the lateral reticular formation: the dorsal aspect of the nucleus reticularis parvocellularis and its caudal continuation, the nucleus reticularis dorsalis. Fewer projections emerged from two nuclei of the medial reticular formation. The dorsal part of the nucleus reticularis ventralis at the spinomedullary junction contributed bilateral with mainly contralateral input to hypoglossal neurons. A few labeled neurons were situated bilaterally in the nucleus reticularis gigantocellularis of the rostral medulla. The input from the spinal V complex originated from the dorsal aspect along most of its length but particularly from the pars interpolaris and oralis subdivisions. Labeled neurons were located primarily in the posterior portion of the nucleus of the solitary tract. Projections from the spinal V complex and the solitary nucleus exhibited ipsilateral predominance. These results suggest that somatic and visceral centers of the rat brainstem play an important role in the control of the activity of hypoglossal motoneurons.