Recent studies have provided evidence that sulfonylureas, in addition to blocking ATP-sensitive K⁺ (K_ATP) channels, also inhibit cystic fibrosis transmembrane regulator (CFTR) Cl⁻ channels in epithelial and cardiac cells. The purpose of this study was to test whether the sulfonylurea glibenclamide might also inhibit other types of cardiac Cl⁻ channels. Whole-cell patch-clamp techniques were used to compare the effects of glibenclamide on CFTR Cl⁻ currents in guinea pig ventricular myocytes, swelling-activated Cl⁻ currents in guinea pig atrial myocytes, and Ca²⁺-activated Cl⁻ currents in canine ventricular myocytes. Glibenclamide markedly inhibited CFTR Cl⁻ currents in a voltage-independent manner at 22°C, with estimated IC₅₀ values of 12.5 and 11.0 μmol/L at +50 and −100 mV, respectively. The outwardly rectifying swelling-activated Cl⁻ current in atrial cells was less sensitive to glibenclamide, and the block exhibited voltage dependence. At 22°C, the estimated IC₅₀ values were 193 and 470 μmol/L at +50 and −100 mV, respectively, and block was enhanced at 35°C. Macroscopic Cl⁻ currents activated by a rise in intracellular Ca²⁺, induced by either Ca²⁺-induced Ca²⁺ release or by external application of the Ca²⁺ ionophore A23187, were also markedly inhibited at 22°C by glibenclamide in a voltage-independent manner. The estimated IC₅₀ values were 61.5 and 69.9 μmol/L at +50 and −100 mV, respectively. These results suggest that glibenclamide, an inhibitor of cardiac CFTR Cl⁻ channels, also inhibits swelling-activated and Ca²⁺-activated Cl⁻ channels at higher concentrations. The results also suggest that studies attributing the beneficial or deleterious effects of sulfonylurea compounds in the heart solely to blockade of K_ATP channels should use submicromolar concentrations of these agents to minimize possible secondary interactions with cardiac Cl⁻ channels.