Monoamines, such as serotonin, dopamine, and norepinephrine, are sequestered into synaptic vesicles by specific transporters (vesicular monoamine transporter-2; VMAT2) using energy from an electrochemical proton gradient across the vesicle membranes. Based on our previous studies using photoaffinity-labeling techniques in characterizing the VMAT2-specific ligands ketanserin and tetrabenazine, this study describes the synthesis and characterization of a fluorenone-based compound, iodoaminoflisopolol (IAmF), as a photoprobe to identify the substrate binding site(s) of VMAT2. Using vesicles prepared from rat VMAT2 containing recombinant baculovirus-infected Sf9 cells, we show the inhibition of [3H]5-hydroxytryptamine (5-HT) uptake and [3H]dihydrotetrabenazine (TBZOH) binding by aminoflisopolol and iodoaminoflisopolol. The interaction of [125I]IAmF with VMAT2 is highly dependent on the presence of ATP and an intact proton gradient. We report a simple and novel method to distinguish between a ligand and substrate using classic compounds such as [3H]5-HT and [3H]TBZOH by incubating the compound with the vesicles followed by washes with isotonic and hypotonic solutions. Using this method, we confirm the characterization of IAmF as a novel VMAT2 substrate. Sf9 vesicles expressing VMAT2 show reserpine- and tetrabenazine-protectable photolabeling by [125I]IAmF. [125I]IAmF photolabeling of recombinant VMAT2, expressed in SH-SY5Y cells with an engineered thrombin site between transmembranes 6 and 7, followed by thrombin digestion, retained photolabel in a 22-kDa fragment, indicating that iodoaminoflisopolol binds to the C-terminal half of the VMAT2 molecule. Thus, IAmF possesses a unique combination of VMAT2 substrate properties and a photoprobe and is, therefore, useful to identify the substrate binding site of the vesicular transporter.