The distribution of GABAergic and glycinergic premotor neurons projecting to the trigeminal motor nucleus (Vm) was examined in the lower brainstem of the rat by a double labeling method combining retrograde axonal tracing with immunofluorescence histochemistry. After injection of the fluorescent retrograde tracer, tetramethylrhodamine dextran amine (TRDA), into the Vm unilaterally, neurons labeled with TRDA were seen ipsilaterally in the mesencephalic trigeminal nucleus, and bilaterally in the parabrachial region, the supratrigeminal and intertrigeminal regions, the reticular formation just medial to the Vm, the principal sensory and spinal trigeminal nuclei, the pontine and medullary reticular formation, especially the parvicellular part of the medullary reticular formation, the alpha part of the gigantocellular reticular nucleus, and the medullary raphe nuclei. Some of these neurons labeled with TRDA were found to display glutamic acid decarboxylase (the enzyme involved in GABA synthesis)-like or glycine-like immunoreactivity. Such double-labeled neurons were seen mainly in the supratrigeminal region, the reticular region adjacent to the medial border of the Vm, and the dorsal part of the lateral reticular formation of the medulla oblongata; a number of them were further scattered in the intertrigeminal region, the alpha part of the gigantocellular reticular nucleus, the nucleus raphe magnus, the principal sensory trigeminal nucleus, and the interpolar subnucleus of the spinal trigeminal nucleus. These neurons were considered to be inhibitory (GABAergic or glycinergic) neurons sending their axons to motoneurons in the Vm, or to local interneurons within and around the Vm.