In order to identify cytochemical traits relevant to understanding excitatory neurotransmission in brainstem auditory nuclei, we have analyzed in the dorsal cochlear nucleus the synaptic distribution of aspartate aminotransferase, glutamate, and vesicular zinc, three molecules probably involved in different steps of excitatory glutamatergic signaling. High levels of glutamate immunolabeling were found in three classes of synaptic endings in the dorsal cochlear nucleus, as determined by quantitation of immunogold labeling. The first type included auditory nerve endings, the second were granule cell endings in the molecular layer, and the third very large endings, better described as "mossy." This finding points to a neurotransmitter role for glutamate in at least three synaptic populations in the dorsal cochlear nucleus. The same three types of endings enriched in glutamate immunoreactivity also contained histochemically detectable levels of aspartate aminotransferase activity, suggesting that this enzyme may be involved in the synaptic handling of glutamate in excitatory endings in the dorsal cochlear nucleus. There was also extrasynaptic localization of the enzyme. Zinc ions were localized exclusively in granule cell endings, as determined by a Danscher-selenite method, suggesting that this ion is involved in the operation of granule cell synapses in the dorsal cochlear nucleus.