Aim: To trace the origin of abundant vesicular glutamate transporter 1-like immu-noreactive (VGIuT1-LI) axon terminals in the dorsal division of the principal sensory trigeminal nucleus (Vpd) and the relationships between VGIuT1-LI, as well as the glutamic acid decarboxylase (GAD)-LI axon terminals, and phosphate-activated glutaminase (PAG)-LI thalamic projecting neurons in the Vpd. Methods: Following unilateral trigeminal rhizotomy, triple-immunofluorescence histoche-mistry for VGluT1, GAD and PAG and the immunogold-silver method for VGIuT1or GAD, combined with the immunoperoxidase method for PAG were performed, respectively. Results: After unilateral trigeminal rhizotomy, the density of VGluT1-like immunoreactivity (IR) in the Vpd on the lesion side was reduced compared to its contralateral counterpart. Under the confocal laser-scanning microscope, theVGIuT1-LI or GAD-LI axon terminals were observed to be in close apposition to the PAG-LI thalamic projecting neuronal profiles, and further electron microscope immunocytochemistry confirmed that VGluT1- and GAD-LI axon terminals made asymmetrical and symmetrical synapses upon the PAG-LI neuronal structures. Conclusion: The present results suggest that the VGluT1-LI axon terminals, which mainly arise from the primary afferents of the trigeminal ganglion, along with the PAG-LI neuronal profiles, form the key synaptic connection involved in sensory signaling.