Evidence that glutamate acts as a neurotransmitter in vestibular nerve fibers was sought (1) by electron microscope radioautographic identification of the uptake sites of [3H]-glutamic acid after incubation of slices of cat vestibular nuclei, and (2) by measuring changes in sodium-dependent high affinity glutamate uptake in nerve endings containing homogenates from normal and deafferented vestibular nuclei 8 to 11 days after unilateral vestibular nerve lesion. Electron microscopic radioautography revealed that glutamate had been taken up by numerous nerve endings projecting over the whole vestibular nuclear complex. The biochemical approach indicated that after section of the vestibular nerve, a significant decrease in high affinity glutamate uptake occurred in the vestibular nuclei, which lost their exclusively ipsilateral projection. This decrease varied from one area of the deafferented vestibular nuclei to another, reaching -58% in the lateral area of the central part corresponding to the ventral lateral vestibular nucleus and the rostral part of the descending vestibular nucleus. It is concluded that glutamate (or aspartate) is used by the vestibular nerve fibers as a neurotransmitter in the vestibular nuclei.