Trigeminothalamic projection neurons are important components of the pathways for conscious perception of pain, temperature, and tactile sensation from the orofacial region. The neurotransmitters utilized by trigeminal neurons projecting to the thalamus are unknown. By use of a monoclonal antibody specific for fixative-modified glutamate and a polyclonal antiserum against glutaminase, we recently identified neurons in the trigeminal sensory complex that contain glutamate-like immunoreactivity (Glu-LI) and glutaminase-like immunoreactivity. In the present study, we utilized combined retrograde transport-immunohistochemical techniques to localize putative glutamatergic trigeminothalamic neurons. Following injection of the retrograde tracer, wheatgerm agglutinin conjugated to horseradish peroxidase (WGA:HRP), into the ventroposterior medial thalamus (VPM), the number of neuronal profiles that were double-labeled with WGA:HRP and Glu-LI was greatest in principal sensory nucleus (Pr5), followed by subnuclei interpolaris (Sp5I) and caudalis (Sp5C). The average percentages of projection neurons double-labeled with Glu-LI were approximately 60-70% in Pr5 and Sp5I and 40% in Sp5C. The majority of double-labeled profiles in Sp5C were located in the magnocellular layer, as opposed to the marginal and substantia gelatinosa layers. A large injection site that spread into the intralaminar thalamic nuclei and nucleus submedius--areas implicated in the processing of nociceptive information--resulted in an increase in the ratio of single-labeled to double-labeled projection profiles in Sp5C. These results suggest that glutamate may be the neurotransmitter for a majority of trigeminothalamic projection neurons located in Sp5I and Pr5. However, on the basis of anatomical association, glutamate does not appear to be the major transmitter for neurons in Sp5C that forward nociceptive information to the thalamus.