To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion: (a) inhibits epibatidine-induced Ca 2+ influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with 100-fold higher potency compared to that in the open state, (c) increases desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits [ 3 H]TCP and [ 3 H]imipramine binding to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/α-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy-driven process, and (f) interacts with a binding domain located between the serine (position 6') and valine (position 13') rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process.Bupropion is an antidepressant that is also being marketed as an aid to smoking cessation (1, 2). The proposed mechanism of action for bupropion is that this drug inhibits the catecholamine reuptake in presynaptic neurons, modulating the concentrations of dopamine and norepinephrine in the synaptic cleft. However, the affinity of bupropion for the neurotransmitter transporter is only moderate, and there is not clear-cut evidence explaining the dual antidepressant and anti-nicotinic modes of action elicited by bupropion.In addition to the current clinical uses of bupropion, this drug behaves pharmacologically as a noncompetitive antagonist (NCA) 1 on several nicotinic acetylcholine receptors (AChRs) (3, † This research was supported by grants from the Science Foundation Arizona and Stardust Foundation and the Office of Research and Sponsored Programs, Midwestern University (to H.R.), by grants from ANPCyT, CONICET, UNS, Loreal-UNESCO, and Fundación F. Fiorini (to C.B), by the FOCUS research subsidy from the Foundation for Polish Science (to K.J.). This research was also supported in part by the Intramural Research Program of the NIH, National Institute on Aging.*To whom correspondence should be addressed: Department of Pharmaceutical Sciences, College of Pharmacy, Midwestern University, 19555 N. 59 th Ave., Glendale, AZ 85308, USA. Telephone: (623) (5)]. AChRs are the paradigm of the Cys-loop ligand-gated ion channel superfamily. This genetically-linked superfamily includes types A and C γ-aminobutyric acid, type 3 5-hydroxytryptamine (serotonin), and glycine receptors [revie...