We previously found evidence for the exocytosis of aspartate from excitatory nerve terminals in hippocampus [J. Neurosci. 18, (1998) 6059]. Here we show, by immunogold electron microscopy in hippocampal slices that aspartate is co-localized and co-exocytosed with GABA from synaptic vesicles in nerve endings assumed to be inhibitory on dentate granule cells and CA1 pyramidal cells. By immunogold double labeling cytochemistry in perfusion fixed hippocampus, we further find that GABA-positive terminals forming symmetric synaptic specializations on perikarya of granule and pyramidal cells express NMDA receptors. In addition, NMDA receptors are present at synapses on granule cell bodies that have asymmetric synaptic specializations and contain high levels of GABA. Glutamate levels are low in the described types of GABA-positive nerve terminals, but high in terminals making asymmetric synapses on dendritic spines, whereas aspartate is localized with high levels in all of these types of terminal. We propose that aspartate is exocytotically released not only from glutamatergic terminals, but also from GABAergic terminals to act on NMDA receptors and may have a role in the regulation of synaptic transmission. Under pathological conditions, release of an excitatory transmitter at an inhibitory synapse could contribute to the development of, for example, epilepsy.