Electrophysiological studies have suggested that inhibition of trigeminal motoneurons during mastication and the jaw-opening reflex are mediated by last-order interneurons (premotoneurons) utilizing GABA and glycine [Chandler et al. (1985), Brain Res., 325:181-186; Enomoto et al. (1987), Neurosci. Res., 4:396-412; Goldberg and Nakamura (1968), Experientia, 24:371-373; Kidokoro et al. (1968), J. Neurophysiol., 31:695-708; Nakamura et al. (1978), Exp. Neurol., 61:1-14]. In the present study we performed a series of double-labeling experiments in guinea pigs to determine the location of neurons which contain GABA (gamma aminobutyric acid) or glycine that project to the trigeminal motor nucleus (Mo5). This was accomplished by performing immunohistochemical staining in combination with a retrograde tract tracing technique using colloidal gold bound to inactivated WGA-HRP (wheat germ agglutin-horseradish peroxidase) (gWGA-HRP) as our retrograde tracer. Neurons which had a positive immunoreactivity to GABA or GAD (glutamic acid decarboxylase) and contained the retrograde marker were located in regions adjacent to the Mo5 such as the intertrigeminal, supratrigeminal, peritrigeminal and rostral portions of the parvocellular reticular formation alpha. Neurons which had a positive immunoreactivity to glycine and contained the retrograde marker were identified in the parvocellular reticular formation, the spinal trigeminal nucleus oralis, supratrigeminal and intertrigeminal regions. These data provide anatomical evidence for GABAergic and glycinergic projections to Mo5.