Loreclezole is a newly developed antiepileptic drug which has been shown to act at a specific site on beta 2 or beta 3 GABAA receptor subtypes to enhance the peak whole-cell response to submaximal concentrations of GABA. Potentiation by loreclezole occurred with high affinity only at GABAA receptors containing a beta 2 or beta 3 subtype, not a beta 1 subtype. We have studied the effect of loreclezole on whole-cell currents from recombinant GABAA receptors transiently expressed in L929 fibroblasts and on currents from cultured mouse cortical neurons and have found a second, inhibitory action of loreclezole that was independent of the beta-subunit subtype composition of the receptor. Loreclezole, at concentrations above 6 microM, enhanced the degree and rate of apparent desensitization of the whole-cell current in a concentration-dependent manner. This effect was voltage-independent, non-competitive and increased with increasing GABA concentration. The increase in desensitization was not blocked by the benzodiazepine antagonist flumazenil and did not require the presence of a gamma subunit. Loreclezole acted at a novel inhibitory allosteric site to increase the apparent desensitization of the GABAA receptor, regardless of its subunit composition. This activity of loreclezole may have implications for its experimental or clinical use as an antiepileptic drug.