Previous studies have indicated that the geniohyoid (GH) muscle is innervated by efferent axons from both the hypoglossal nerve (CN XII) and ansa cervicalis. To clarify the physiological significance of this dual innervation of the GH muscle, we examined properties of efferent innervations in rat GH muscle using electrophysiological, horseradish peroxidase (HRP) tracing and immunohistochemical techniques. Recordings from the branch of the XII nerve that innervates the GH (GH.Br) revealed that bursts of impulses during fictitious swallowing were conducted via the XII nerve trunk, in which neuronal cell bodies were labeled in the ventrolateral subnucleus of the XII nucleus by HRP tracing. In contrast, in vivo experiments demonstrated that tonic discharges in GH.Br were conducted via the ansa cervicalis. However, HRP-labeled efferent neurons were observed in neither brainstem nor upper spinal cord, but sensory neurons were labeled in the most rostral cervical spinal ganglia via the ansa cervicalis. Tonic activity was abolished in vitro by mecamylamine, an antagonist of nicotinic acetylcholine receptors (nAChR), and by pirenzepine, an antagonist of muscarinic M1 receptors. Incubation of isolated XII nerve segments with antisera to vasoactive intestinal peptide, nAChR, and muscarinic M1 receptor yielded small numbers of labeled neurons with each antiserum. All labeled neurons displayed similar diameters and were located approximately 1.5 mm proximal to the bifurcation of the XII nerve into medial and lateral branches. Our findings indicate that GH muscle in the rat is innervated by both somatic and parasympathetic nervous systems.