Acetylcholine (ACh), the first neurotransmitter ever to be identified, is a small- molecule excitatory neurotransmitter with a wide variety of known functions. Within the central nervous system, cholinergic cells (neurons that use ACh as a neurotransmitter) are found in several different locations of the brain, including the striatal complex, the basal forebrain, the diencephalon, pontomesencephalic cell groups, and the medulla. Depending on which area of the brain it is found in, ACh may be involved in any one of several different functions. Some of these functions include the conduction of pain, the regulation of neuroendocrine function, the regulation of REM sleep cycles, and the process of learning and memory formation. In the sympathetic and parasympathetic nervous systems and at all neuromuscular junctions, ACh is used to signal muscle movement.

Whether it is within the brain, or at the neuromuscular junction, when ACh is released into the synaptic cleft, it transiently binds with a ligand-gated receptor molecule on the post-synaptic neuron or muscle cell. This binding causes the receptor molecule to open up, allowing for an exchange of ions which subsequently results in a depolarization of the post-synaptic neuron. If this depolarization is strong enough, an action potential is stimulated.

After depolarization, the neurotransmitter must be swiftly removed from the receptor molecule and inactivated in order to prevent constant stimulation of the post-synaptic membrane and an excessive firing of action potentials. ACh is one of several neurotransmitters whose action at the synaptic cleft is terminated by the hydrolyizing action of an enzyme. Once ACh has been recognized and the post-synaptic membrane has been depolarized, ACh quickly dissociates from the receptor and binds to acetylcholinesterase (AChE), an enzyme named for its ability to break the ester bond that holds acetate and choline together. Removal of ACh from the recognition site causes the membrane-spanning pore of the receptor to close. The electric potential of the membrane then normalizes and the action potential ceases to fire. The breakdown of ACh by AChE inactivates the neurotransmitter and prevents it from reassociating with the receptor molecule.