The potential drug target choline acetyltransferase (ChAT) catalyzes the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells . Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction . This in-situ synthesis yields an adduct that is the actual enzyme inhibitor . The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors . Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.