The spinal trigeminal nucleus (STN) is involved in processing orofacial sensory information, including tactile, thermal and nociceptive input, and relaying this information to higher brain centers, such as the thalamus. Very little information is available regarding the major excitatory neurotransmitters of this nucleus. The amino acid glutamate has been proposed as a major excitatory neurotransmitter in the central nervous system. In the present study, a novel monoclonal antibody, specific for fixative-modified glutamate, was utilized in conjunction with polyclonal antisera against glutaminase and aspartate aminotransferase (AATase) in an attempt to identify and map the locations of possible glutamatergic neurons in the STN. Co-localization experiments were performed by radiolabeling our monoclonal antibody and using this antibody in conjunction with the polyclonal antisera against glutaminase and AATase to evaluate the possible coexistence of glutamate with glutaminase or AATase in STN neurons. In all three subnuclei of the STN, immunohistochemically labeled neuronal profiles were observed with both of the polyclonal antisera and with the monoclonal antibody. Subnucleus caudalis contained the greatest number of labeled profiles per coronal section followed by subnucleus interpolaris and subnucleus oralis. The number and the distribution of immunoreactive profiles observed after the use of the glutaminase antiserum was comparable to that obtained with the monoclonal antibody. Co-localization experiments demonstrated that all glutaminase-like immunoreactive neurons also contained fixative-modified glutamate-like immunoradioactivity. These results suggest that glutamatergic neurons are present in the spinal trigeminal nucleus. The AATase antiserum labeled more neuronal profiles in each of the three subnuclei than did the glutaminase antiserum or the monoclonal antibody. In addition, co-localization experiments indicated that glutamate-like immunoreactivity was present in only two-thirds of AATase-like immunoreactive neuronal profiles. These findings suggest that glutaminase may be a more reliable marker of glutamatergic function than AATase.