There is currently great interest in developing radiolabeled substrates for acetylcholinesterase that would be useful in the in vivo imaging of patients with Alzheimer's disease. The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using 11C and 18 F-labeled acetylcholine analogues. Our aim was to develop a new 99mTc-labeled acetylcholine analogue: N-phenylferrocenecarboxamide labelled with technetium-99 m (99mTc-TPCC) to study acetylcholinesterase activity. In vivo and in vitro studies demonstrated that the labelled compound was a substrate for acetylcholinesterase. The hydrolytic rate of this substrate was measured and the specificity was evaluated using the inhibitor BW 284 C51. In rat experiments, the 99mTc-TPCC showed desirable properties for studying the acetylcholinesterase in the rat brain: high hydrolytic rate and a moderate specificity of the substrate for acetylcholinesterase.