To examine the possible roles of glutamate and aspartate as neurotransmitters in the nucleus submedius (Sm) of rats, the distributions of these amino acids were examined by electron microscopic immunogold labeling. High levels of glutamate were detected in trigeminothalamic tract terminals anterogradely labeled with horseradish peroxidase conjugates. These terminals also displayed a positive correlation between the densities of synaptic vesicles and gold particles signaling glutamate. In contrast, aspartate levels in such terminals were low and displayed no correlation with the density of synaptic vesicles. Terminals of presumed cortical origin contained the highest estimated levels of glutamate, but the positive correlation between glutamate signal and synaptic vesicle density did not reach statistical significance, presumably due to technical factors. The latter terminals also contained relatively high levels of aspartate, though without any correlation to synaptic vesicle density. The present findings provide strong support for glutamate, but not aspartate, as a trigeminothalamic tract neurotransmitter responsible for the fast synaptic transmission of nociceptive signals to neurons in the rat nucleus submedius. Aspartate presumably serves metabolic roles in these terminals. With respect to terminals of presumed cortical origin, our data are not at odds with the notion that also these terminals use glutamate as their neurotransmitter. Our findings do not support a neurotransmitter role for aspartate in the latter terminals, although such a role cannot be entirely refuted.