Regional differences in cholinergic activity in the cardiac conduction system have been difficult to study. We tested the utility of (+)-m-[125I]iodobenzyl)trozamicol(+)-[125I]MIBT), a novel radioligand that binds to the vesamicol receptor located on the synaptic vesicle in presynaptic cholinergic neurons, as a functional marker of cholinergic activity in the conduction system. The (+)-[125I]MIBT was injected intravenously into four rats. Three hours later, the rats were killed and their hearts were frozen. Quantitative autoradiography was performed on 20-micron-thick sections that were subsequently stained for acetylcholinesterase to identify specific conduction-system elements. Marked similarities existed between (+)-[125I]MIBT uptake and acetylcholinesterase-positive regions. Optical densitometric analysis of regional (+)-[125I]MIBT uptake revealed significantly greater (+)-[125I]MIBT binding (nCi/mg) in the atrioventricular node (AVN) and His bundle regions compared with other conduction and contractile elements (AVN: 3.43 +/- 0.37; His bundle: 2.16 +/- 0.30; right bundle branch: 0.95 +/- 0.13; right atrium: 0.68 +/- 0.05; right ventricle: 0.57 +/- 0.03; and left ventricle: 0.57 +/- 0.03; p < 0.05 comparing conduction elements with ventricular muscle). This study demonstrates that (+)-[125I]MIBT binds avidly to cholinergic nerve tissue innervating specific conduction-system elements. Thus, (+)-[125I]MIBT may be a useful functional marker in studies on cholinergic innervation in the cardiac conduction system.