Glybenclamide is used as a pharmacological tool in studies of mitochondrial functions supposing its main role to block ATP-dependent potassium (K-ATP) channel. The aim of this study was to test whether glybenclamide might interact with the mitochondrial chloride channels. Mitochondrial membranes, isolated from rat heart muscle, were incorporated into lipid bilayer membrane and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. The observed chloride channels (N = 11) with mean conductance 120 +/- 14 pS were sensitive to glybenclamide, which decreased the open probability (IC50 = 129 mu M) and affected the channel gating kinetics (IC50 = 12 mu M) by perturbing its open state. It did not influence the channel conductance or reversal potential. These results indicate that glybenclamide interacts with chloride channels what should be taken into consideration, when glybenclamide is used as a specific inhibitor of K-ATP channels. (C) 2013 Elsevier Inc. All rights reserved.