A number of cyclic quaternary ammonium derivatives structurally related to acetylcholine were tested for their inhibitory effect on choline acetyltransferasein vitro.They had no effect. The alcohol derivatives in this series of compounds were not acetylated by the enzyme. The tertiary amine 2‐dimethylaminoethyl chloroacetate and a few related compounds had inhibitory activityin vitro.2‐Dimethylaminoethyl chloroacetate was the most active of these compounds, being about half as potent as the quaternary derivative choline chloroacetate. It inhibited the enzyme uncompetitively with regard to choline as well as acetyl‐CoA, the Kivalues being 1.2 × 10‐4M (choline) and 4 × 10‐5M (acetyl‐CoA) under the conditions used. 2‐Dimethylaminoethyl chloroacetate and two of the other active compounds had no effect in high doses on the acetylcholine level in the mou