Aspartate can be released from certain hippocampal pathways along with glutamate or GABA. Although aspartate immunoreactivity has been localized to synaptic vesicles and aspartate release is Ca(2+)-dependent, there has been no clear evidence favoring an exocytotic mechanism. In particular, pretreatment with Clostridial toxins has not consistently inhibited aspartate release, even when release of glutamate from the same tissue samples was markedly inhibited. To address this issue directly, rat hippocampal synaptosomes were permeabilized transiently by electroporation in the presence of active or inactivated Clostridial toxin light chains. Loading rat hippocampal synaptosomes with the active light chain of tetanus toxin or of botulinum neurotoxins A, B or C reduced the K(+)-evoked release of aspartate at least as much as that of glutamate. These results confirm that aspartate is released by exocytosis in rat hippocampus.