The acetate required for the synthesis of ACh comes from Acetyl CoenzymeA. Coenzyme A (CoA) forms a covalent bond with the acyl group in acetate and thus serves as a carrier of the acetate. Acetyl CoA is synthesized in the mitochondria of all cells as a by-product of the pyruvate dehydrogenase complex. Much of the Acetyl CoA produced by this process is used to generate ATP in the Krebs cycle. However, in cholinergic cells, some of this acetyl CoA is transported across the mitochondrial membrane into the cytoplasm for use in the synthesis of ACh.

Coenzyme A remains bonded to the acyl group of acetate until it reacts with choline to form ACh. This simple, one-step reaction requires the enzyme choline acetyltransferase (CAT). CAT is synthesized in the rough endoplasmic reticulum of all cholinergic cells and then transported by slow axoplasmic transport to the axon terminal, where it is readily available for the synthesis of ACh.

It is thought that acetylcholine synthesis begins with CAT first forming a complex with acetyl CoA. Next, choline binds to an adjacent site on the enzyme. After both precursors are bound, choline is transferred to acetyl CoA and the two molecules form a complex. CAT and coenzyme A are then released, and what remains is acetylcholine.