A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.