A fresh brain-slice preparation that allows simultaneous characterization of the anatomic localization of radioactively labeled substances in a variety of different auditory brain stem areas has been used to examine localization patterns for a number of putative neurotransmitter amino acids in the anteroventral and dorsal cochlear nuclei of the cat. Following incubation of fresh brain sections in oxygenated salt solutions containing micromolar amounts of the tritiated amino acids, differences in the pattern of label localization are revealed by light microscopic autoradiography. Electron microscopic autoradiography of the same material demonstrates that the label is restricted to different synaptic terminal populations. In the dorsal cochlear nucleus, gamma aminobutyric acid (GABA) and glutamic acid incubations label two different terminal populations in the molecular layer. Glycine incubation labels a population more uniformly distributed among the molecular, fusiform cell, and deep layers. In the anteroventral cochlear nucleus the pattern after GABA and glycine incubations is similar, with multiple endings on all spherical cells labeled. Studies are underway to characterize fully the different labeling patterns and the conditions under which they occur.