Glutamate and aspartate are known to be released in a calcium-dependent fashion by depolarizing stimulation of mammalian brain synaptosomes (isolated nerve endings), an observation which strengthens their claims to be neurotransmitter candidates. The source of these compounds has been interpreted as the exclusively glutamatergic or aspartatergic synaptosome sub-populations assumed to be present in the standard heterogeneous preparations from mammalian brain. Several neurotransmitter-specific synaptosomal surface markers have recently been identified by immunolysis studies and these have allowed separation of subpopulations of synaptosomes by an affinity purification method. These markers appear to be closely related to the biosynthetic enzyme for the principal neurotransmitter released by each sub-category of synaptosome. We have isolated highly purified, metabolically active, GABAergic and cholinergic synaptosomes from cerebral cortex using antisera recognizing either glutamate decarboxylase (GAD) or choline acetyltransferase (ChAT), in conjunction with magnetic microspheres covalently coupled to Protein A (ref. 8), and now report that these synaptosomes release both glutamate and aspartate, in addition to their principal neurotransmitter, when treated with chemical depolarizing agents.