The role of amino acid neurotransmitters in learning and memory is well established. We investigated the putative role of L-aspartate as a neurotransmitter in the arcopallial-medial striatal pathway, which is known to be involved in passive avoidance learning in domestic chicks. Double immunocytochemistry against L-aspartate and L-glutamate was performed at both light and electron microscopic levels. L-aspartate- and L-glutamate-immunoreactive neurons in the arcopallium and posterior amygdaloid pallium were identified and counted by using fluorescence microscopy and confocal laser scanning microscopy. Most labeled neurons of arcopallium were enriched in glutamate as well as aspartate. However, the arcopallium and posterior amygdaloid pallium differed from a neighboring telencephalic region (nidopallium; formerly neostriatum) by containing a substantial proportion of cells singly labeled for L-aspartate (15%, vs. 5.3% in the nidopallium). Aspartate-labeled neurons constitute approximately 20%, 25%, 42%, and 28% of total in the posterior amygdaloid pallium and the medial, dorsal, and anterior arcopallia, respectively. Immunoelectron microscopy showed that L-aspartate was enriched in terminals of the medial striatum. The labeled terminals had clear and round vesicles and asymmetric junctions; similar to those immunoreactive to L-glutamate. Axon terminals singly labeled for L-aspartate made up 17% of the total. In addition, 7% of neuronal perikarya and 26% of all dendritic profiles appeared to be labeled specifically with L-aspartate but not L-glutamate. The results indicate that L-aspartate may play a specific role (as distinct from that of L-glutamate) in the intrinsic and extrinsic circuits instrumental in avian learning and memory.