To determine whether (+)-catharanthine induces sedative- or anxiolytic/anxiogenic-like activity in male mice, proper animal paradigms were used. The results showed that (+)-catharanthine induces sedative-like activity in the 63-72 mg/Kg dose range in a flumazenil-insensitive manner, but neither this effect nor anxiolytic/anxiogenic-like activity was observed at lower doses. To determine the underlying molecular mechanism of the sedative-like activity, electrophysiological and radioligand binding experiments were performed with (+)-catharanthine and (+/-)-18-methoxycoronaridine [(+/-)-18-MC] on GABA(A) (GABA(A)Rs) and glycine receptors (GlyRs). Coronaridine congeners both activated and potentiated a variety of human (h) GABA(A)Rs, except hrho1. (+)-Catharanthine-induced potentiation followed this receptor selectivity (EC(50)'s in muM): halpha1beta2 (4.6 +/- 0.8) > halpha2beta2gamma2 (12.6 +/- 3.8) ~ halpha1beta2gamma2 (14.4 +/- 4.6) indicating that both alpha1 and alpha2 are equally important, whereas gamma2 is not necessary. (+)-Catharanthine was >2-fold more potent and efficient than (+/-)-18-MC at halpha1beta2gamma2. (+)-Catharanthine also potentiated, whereas (+/-)-18-MC inhibited, halpha1 GlyRs with very low potency. Additional [(3)H]-flunitrazepam competition binding experiments using rat cerebellum membranes clearly demonstrated that these ligands do not bind to the benzodiazepine site. This is supported by the observed activity at halpha1beta2 (lacking the BDZ site) and similar effects between alpha1- and alpha2-containing GABA(A)Rs. Our study shows, for the first time, that (+)-catharanthine induced sedative-like effects in mice, and coronaridine congeners potentiated human alpha1beta2gamma2, alpha1beta2, and halpha2beta2gamma2, but not rho1, GABA(A)Rs, both in a benzodiazepine-insensitive fashion, whereas only (+)-catharanthine slightly potentiated GlyRs. CI - Copyright (c) 2020 Elsevier Inc. All rights reserved.