Amino acids have important roles in the chemistry of the auditory system, including communication among neurons. There is much evidence for glutamate as a neurotransmitter from auditory nerve fibers to cochlear nucleus neurons. Previous studies in rodents have examined effects of removal of auditory nerve input by cochlear ablation on levels, uptake and release of glutamate in cochlear nucleus subdivisions, as well as on glutamate receptors. Effects have also been reported on uptake and release of γ-aminobutyrate (GABA) and glycine, two other amino acids strongly implicated in cochlear nucleus synaptic transmission. We mapped the effects of cochlear ablation on the levels of amino acids, including glutamate, GABA, glycine, aspartate, glutamine, taurine, serine, threonine, and arginine, in microscopic subregions of the rat cochlear nucleus. Submicrogram-size samples microdissected from freeze-dried brainstem sections were assayed for amino acid levels by high performance liquid chromatography. After cochlear ablation, glutamate and aspartate levels decreased by 2 days in regions receiving relatively dense innervation from the auditory nerve, whereas the levels of most other amino acids increased. The results are consistent with a close association of glutamate and aspartate with auditory nerve fibers and of other amino acids with other neurons and glia in the cochlear nucleus. A consistent decrease of GABA level in the lateral superior olive could be consistent with a role in some lateral olivocochlear neurons. The results are compared with those obtained with the same methods for the rat vestibular nerve root and nuclei after vestibular ganglionectomy.