The discovery of the first neurotransmitter–acetylcholine–was soon followed by the discovery of its hydrolysing enzyme, acetylcholinesterase. The role of acetylcholinesterase in terminating acetylcholine-mediated neurotransmission made it the focus of intense research for much of the past century. But the complexity of acetylcholinesterase gene regulation and recent evidence for some of the long-suspected ‘non-classical’ actions of this enzyme have more recently driven a profound revolution in acetylcholinesterase research. Although our understanding of the additional roles of acetylcholinesterase is incomplete, the time is ripe to summarize the evidence on a remarkable diversity of acetylcholinesterase functions.