Acetylcholinesterase, also known as AChE or acetylhydrolase, is a serine protease that hydrolyzes the neurotransmitter acetylcholine. AChE is found at mainly neuromuscular junctions and cholinergic brain synapses, where its activity serves to terminate synaptic transmission. It belongs to carboxylesterase family of enzymes.

AChE has a very high catalytic activity - each molecule of AChE degrades about 25000 molecules of acetylcholine (ACh) per second, approaching the limit allowed by diffusion of the substrate. The active site of AChE comprises 2 subsites - the anionic site and the esteratic subsite. The structure and mechanism of action of AChE have been elucidated from the crystal structure of the enzyme.

The anionic subsite accommodates the positive quaternary amine of acetylcholine as well as other cationic substrates and inhibitors. The cationic substrates are not bound by a negatively-charged amino acid in the anionic site, but by interaction of 14 aromatic residues that line the gorge leading to the active site. All 14 amino acids in the aromatic gorge are highly conserved across different species. Among the aromatic amino acids, tryptophan 84 is critical and its substitution with alanine results in a 3000-fold decrease in reactivity. The gorge penetrates half way through the enzyme and is approximately 20 angstroms long. The active site is located 4 angstroms from the bottom of the molecule.