The rate at which the transmitter release machinery is fixed by 2% glutaraldehyde at hippocampal synapses and the amount of release evoked by the fixative have been investigated. We recorded from hippocampal cells while fixative was applied with a rapid flow system. Release is blocked in less than a second and fixative-produced exocytosis is at most a few percent of what would be caused by a hypertonic stimulus that completely depletes the readily releasable pool of vesicles. The postsynaptic receptors for glutamate cease to respond to agonist with a time constant of approximately 3 s when fixative is applied. We conclude that some essential component of the exocytotic apparatus is fixed in less than a second and that the fixative does not significantly deplete the readily releasable pool.