D-[3H]aspartate was used to identify potential glutamatergic connections of the chinchilla inferior colliculus (IC). High-affinity uptake of D-[3H]aspartate is considered a selective marker for glutamatergic synapses, and neurons retrogradely labeled from such injections are believed to use glutamate, or a closely related compound, as a transmitter. Injections of D-[3H]aspartate suggest that glutamatergic endings in the IC arise primarily from intrinsic connections, the opposite IC, layer 5 of temporal cortex, nucleus sagulum, and lateral lemniscal nuclei. Neurons giving rise to the principal sensory (lemniscal) projections to the IC, i.e., those from the cochlear nuclei, superior olive, and the majority of projections from the lateral lemniscal nuclei, did not label in these experiments, indicating that their synapses do not recognize D-[3H]aspartate as a suitable substrate and may use inhibitory or other excitatory transmitters. After IC injections, fiber and diffuse labeling was found ipsilaterally in the medial geniculate body, superior colliculus, and dorsolateral pontine nuclei, contralaterally in the IC, and bilaterally in the superior olive and cochlear nuclei. Such labeling was attributed to anterograde transport of D-[3H]aspartate within the efferent collaterals of labeled IC neurons, suggesting that many of the IC's efferent projections may also be glutamatergic. This interpretation was confirmed in separate experiments in which D-[3H]aspartate, injected in the medial geniculate body, retrogradely labeled neurons in the IC as well as in layer 6 of temporal cortex. Finally, the mesencephalic trigeminal nucleus and tract labeled in some cases and may have local glutamatergic connections.