During the past decade, considerable efforts have been made in the development of radiopharmaceuticals for the in vivo study of the cholinergic neurotransmission using positron emission tomography or single photon emission computerized tomography. The main cholinergic radioligands, labelled with positron- or gamma-photon-emitting radionuclides, are reviewed with respect to use as in vivo markers of either acetylcholinesterase, vesicular acetylcholine transporter, brain and heart muscarinic receptors, or cholinergic nicotinic receptors. The main results obtained in the in vivo study of the physiology, pharmacology or pathology of the different steps of the cholinergic neurotransmission using single photon emission computerized tomography and positron emission tomography are discussed.