In the resting state, neurotransmitters are stored in vesicles at the pre-synaptic terminal. Most neurons contain at least two types of synaptic vesicles, small (about 50 nm diameter) and large (70-200 nm diameter). Neurotransmitters are released in quantal units as each vesicle contains a given amount of transmitter. The vesicles are concentrated near "active zones," dense bodies along the pre-synaptic membrane where neurotransmitter release occurs. At rest, only a small number of vesicles are actually positioned at the active zones. Rather, most are held near the active zones by the proteins synapsin I and actin. Synapsin I, a protein associated with the vesicle membrane, binds to actin, a cytoskeletal filament holding the vesicles in place.

Calcium (Ca2+) is a vital element in the process of neurotransmitter release; when Ca2+ channels are blocked, neurotransmitter release is inhibited. When the action potential reaches the nerve terminal, voltage-dependent Ca2+ channels open and Ca2+ rushes into the neuron terminal due to a greater extracellular concentration.

Ca2+ channels appear to be localized near the active zones of the vesicular membrane. In the giant squid neuron, it has been found that Ca2+ influx is ten times greater in the area of the active zone than elsewhere in the neuron. Furthermore, during an action potential Ca2+ concentration at the active zone can rise one-thousandfold, from 100 nM (nano-molar concentration) to 100 micro-M within a few hundred microseconds. The observation of intramembranous proteins thought to be Ca2+ channels near active zones is consistent with rapid neurotransmitter release following Ca2+ influx.