At rest, neurotransmitter-containing vesicles are stored at the terminal of the neuron in one of two places. A small number of vesicles are positioned along the pre-synaptic membrane in places called "active zones." This is where neurotransmitter release occurs. Most vesicles, however, are held close to these zones, yet further from the membrane itself until they are needed. These vesicles are held in place by Ca2+-sensitive vesicle membrane proteins (VAMPs), which bind to actin filaments, microtubules, and various other elements of the cytoskeleton. When an action potential reaches the terminal of a presynaptic neuron, voltage-dependent calcium (Ca2+) channels embedded in the pre-synaptic membrane open and Ca2+ rushes in. This influx of calcium ions triggers a series of events, which ultimately results in the release of the neurotransmitter from a storage vesicle into the synaptic cleft.

The first step in this process involves freeing the neurotransmitter-containing vesicles from the bonds that hold them to the cytoskeleton. The vesicles are then free to travel to the active zones, where docking takes place. Docking is the process by which the vesicle and the pre-synaptic membrane line up in a position that will allow them to fuse easily. Fusion then takes place, in which the vesicle membrane and the pre-synaptic membrane connect to form a small opening, a pore, connecting the lumen of the vesicle with the synaptic cleft. This pore grows larger and larger until the vesicle membrane collapses into the pre-synaptic membrane and releases its contents into the synaptic cleft, a process called exocytosis.

Following exocytosis, the vesicular membrane, presently a continuous extension of the pre-synaptic membrane, forms a pit and pinches off into the terminal to form a new, vacant vesicle. This vesicle is then either recycled and refilled with more of the neurotransmitter, or sent to the cell body, where it is broken down, processed into a new vesicle, and transported to the terminal where it can then be filled with the neurotransmitter.