In this study, the regional rat brain distribution of radioiodinated o-iodo-trans-decalinvesamicol ([125I]OIDV) was determined in vivo to evaluate its potential as a single-photon emission computed tomography (SPECT) imaging probe for vesicular acetylcholine transporter (VAChT). Following intravenous injection, [125I]OIDV passed freely across the blood–brain barrier and accumulated in rat brain. The accumulation of [125I]OIDV in rat brain was significantly reduced by coadministration of (+/−)-vesamicol (0.125 µmol). In contrast, the coadministration of σ-receptor ligands, such as (+)-pentazocine (0.125 µmol) as a σ-1 receptor ligand and (+)−3-(3-hydroxyphenyl)-N-propylpiperidine (0.125 µmol) as a σ-1 and σ-2 receptor ligands, barely affected the accumulation of [125I]OIDV in rat brain. These findings in vivo were corroborated by autoradiographic analysis ex vivo. The authors found that the tracer binds with pharmacological selectivity to VAChT in rat brain and predicted that it may likewise serve in translational SPECT imaging studies of this marker in the integrity of cholinergic innervations. Synapse 68:107–113, 2014. © 2013 Wiley Periodicals, Inc.