Nicotinic acetylcholine receptors (nAChRs) are targeted for drug development for cognitive function, Parkinson's disease, analgesia, and other conditions. Currently, [125I]α-bungarotoxin ([125I]α-BGT) is the only iodine-labeled radioligand specific for the α7 nAChR but has high nonspecific binding in filtration assays and cannot cross the blood-brain barrier. Methyllycaconitine (MLA), isolated from Delphinium brownii seeds, is a highly selective α7 nAChR ligand that is a small, reversible-binding compound able to cross the blood-brain barrier. In this study, we synthesized [125I]iodomethyllycaconitine ([125I]-8) via radioiododestannylation of a trimethylstannyl-MLA precursor (9) using chloramine-T as oxidant, achieving a 74% radiochemical yield after HPLC purification. Competition binding experiments showed the iodo-MLA analogue (8) had affinity for the α7 nAChR almost identical to MLA and poor affinity for α4β2 nAChRs labeled by [3H]epibatidine. Saturation binding experiments in rat brain cerebral cortex homogenates revealed saturable specific binding best fit by a one-site model with an affinity constant (Kd) of 1.8 ± 0.4 nM and a maximal binding capacity (Bmax) of 68 ± 3 fmol/mg protein. Competition binding with known ligands (MLA, α-bungarotoxin, 3-cinnamylidine-anabasine) confirmed [125I]-8's specificity for α7 nAChRs, while nicotine, dihydro-β-erythrodine, and mecamylamine were weak or ineffective competitors. Regional binding studies showed selective [125I]-8 binding in α7-rich brain regions (hippocampus, thalamus/hypothalamus) versus low-binding regions (cerebellum, striatum). In summary, [125I]-8 binds with high specificity to α7 nAChRs, serving as a viable alternative to [125I]α-BGT for studying this subtype. Its high specific activity makes it suitable for high throughput screening assays using brain tissue or cloned receptors, and iodine-123 labeling may enable in vivo imaging of α7 nAChRs.