Nicotinic acetylcholine receptors (nAChRs) containing α6 and β2 subunits modulate dopamine release in the basal ganglia and are therapeutically relevant targets for treatment of neurological and psychiatric disorders including Parkinson's disease and nicotine dependence. However, the expression profile of β2 and β4 subunits overlap in a variety of tissues including locus ceruleus, retina, hippocampus, dorsal root ganglia, and adrenal chromaffin cells. Ligands that bind α6β2 nAChRs also potently bind the closely related α6β4 subtype. To distinguish between these two subtypes, we synthesized novel analogs of a recently described α-conotoxin, PeIA. PeIA is a peptide antagonist that blocks several nAChR subtypes, including α6/α3β2β3 and α6/α3β4 nAChRs, with low nanomolar potency. We systematically mutated PeIA and evaluated the resulting analogs for enhanced potency and/or selectivity for α6/α3β2β3 nAChRs expressed in Xenopus oocytes (α6/α3 is a subunit chimera that contains the N-terminal ligand-binding domain of the α6 subunit). On the basis of these results, second-generation analogs were then synthesized. The final analog, PeIA[S9H,V10A,E14N], potently blocked acetylcholine-gated currents mediated by α6/α3β2β3 and α6/α3β4 nAChRs with IC(50) values of 223 pM and 65 nM, respectively, yielding a >290-fold separation between the two subtypes. Kinetic studies of ligand binding to α6/α3β2β3 nAChRs yielded a k(off) of 0.096 ± 0.001 min(-1) and a k(on) of 0.23 ± 0.019 min(-1) M(-9). The synthesis of PeIA[S9H,V10A,E14N] demonstrates that ligands can be developed to discriminate between α6β2 and α6β4 nAChRs.