Physiological evidence indicates that vestibular signals modulate the activity of motoneurons innervating the masseter muscle. Recently, experiments using transynaptic retrograde transport of pseudorabies virus provided anatomical evidence that many neurons concentrated in the dorsomedial part of the parvicellular division of the medial vestibular nucleus (MVePC) and the caudal prepositus hypoglossi (PH) provide inputs to motoneurons innervating the lower third of the superficial layer of the masseter muscle. However, it was not clear whether this vestibulo-trigeminal projection was monosynaptic or polysynaptic. The present study sought to determine whether neurons in the MVePC or PH project directly to motoneurons controlling the masseter muscle in rats. For this purpose, an anterograde tracer (biotinylated dextran amine, BDA) was injected into vestibular nuclei (mainly MVePC) or PH and a retrograde tracer (the beta-subunit of cholera toxin, b-CT) was injected into the masseter muscle ipsilateral or contralateral to the BDA injection site. Following injections of BDA into the vestibular nuclei or PH, anterogradely labeled axon terminals were observed bilaterally in the motor trigeminal nucleus (Mo5), particularly in the ventral, medial, and lateral portions of the nucleus; projections to dorsal Mo5 were sparse. In addition, retrogradely labeled motoneurons were located in the ventral and lateral portions of the ipsilateral Mo5. Moreover, anterogradely labeled terminals were observed to be in close proximity to motoneurons in the Mo5 that were retrogradely labeled from b-CT injections into the masseter muscle. This study provides direct evidence that a monosynaptic pathway exists between the MVePC and PH and masseter motoneurons.