To determine in rat if vagal afferent fibers projecting into the intermediate one third of the nucleus tractus solitarius (NTS), the site of termination of baroafferents, utilize glutamate as a neurotransmitter, the high-affinity uptake of [3H]L-glutamate and content of glutamate were analyzed in micropunches of rat brain stem. The intermediate NTS contains a high-affinity synaptosomal uptake system for [3H]L-glutamate that is greater in capacity than that in areas adjacent to the NTS; it is almost two-fold higher than uptake in medial septum and nucleus accumbens and equal to that of hippocampal regions purportedly containing a rich glutamatergic innervation. Unilateral ablation of the nodose ganglion (i.e. cells of origin of vagal afferents) resulted, within 24 h in a prolonged significant reduction, to 56% of control, of [3H]L-glutamate uptake, bilaterally in the NTS. The reduction of Na+-dependent synaptosomal uptake of [3H]L-glutamate, resulted from a decrease in Vmax without change in the Km of the process, was anatomically restricted to the intermediate NTS, and was not associated with changes in [3H]GABA uptake. The content of glutamate in the NTS was significantly (P less than 0.01) decreased by 30% 7 days following unilateral extirpation of the nodose ganglion without changes in the concentrations of aspartate, glycine, glutamine, or GABA. A population of vagal afferent fibers projecting to NTS are glutamatergic. The results are consistent with the hypothesis obtained by physiological and pharmacological techniques that glutamate is a neurotransmitter of baroafferents.