The FDA-approved antidepressant and smoking cessation drug bupropion is known to inhibit dopamine and norepinephrine reuptake transporters, as well as nicotinic acetylcholine receptors (nAChRs) which are cation-conducting members of the Cys-loop superfamily of ion channels, and more broadly pentameric ligand-gated ion channels (pLGICs). In the present study, we examined the ability of bupropion and its primary metabolite hydroxybupropion to block the function of cation-selective serotonin type 3A receptors (5-HT(3A)Rs), and further characterized bupropion's pharmacological effects at these receptors. Mouse 5-HT(3A)Rs were heterologously expressed in HEM-293 cells or Xenopus laevis oocytes for equilibrium binding studies. In addition, the latter expression system was utilized for functional studies by employing two-electrode voltage-clamp recordings. Both bupropion and hydroxybupropion inhibited serotonin-gated currents from 5-HT(3A)Rs reversibly and dose-dependently with inhibitory potencies of 87 mu M and 112 mu M, respectively. Notably, the measured IC50 value for hydroxybupropion is within its therapeutically-relevant concentrations. The blockade by bupropion was largely non-competitive and non-use-dependent. Unlike its modulation at cation-selective pLGICs, bupropion displayed no significant inhibition of the function of anion-selective pLGICs. In summary, our results demonstrate allosteric blockade by bupropion of the 5-HT3AR. Importantly, given the possibility that bupropion's major active metabolite may achieve clinically relevant concentrations in the brain, our novel findings delineate a not yet identified pharmacological principle underlying its antidepressant effect. (C) 2016 Elsevier Ltd. All rights reserved.