Neurotransmitter is released when an AP arrives at the axon terminal and causes the opening of voltage-gated Ca²⁺ channels in the presynaptic membrane. Because the Ca²⁺ concentration is greater outside the cell than inside, Ca²⁺ enters the axon terminal. This increase in Ca²⁺ inside the axon terminal causes the vesicles containing neurotransmitter to fuse with the presynaptic membrane and empty their contents into the synaptic cleft.

In neuromuscular synapses, vesicle fusion and emptying is all-or-none. The vesicle membrane is incorporated into the presynaptic membrane, which actually gets larger as a result—at least until the extra membrane is recycled through endocytosis. The membrane is reprocessed by the cell into new vesicles that are refilled with neurotransmitter.