To study the mechanisms of glibenclamide actions on volume-sensitive Cl- channels, whole cell patch-clamp studies were performed at various pH levels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl- currents in the entire range of voltage examined (-100 to +100 mV) and accelerated the depolarization-induced inactivation at pH 7.5. When glibenclamide was applied from the intracellular side, in contrast, no effect was observed. At acidic pH, at which the weak acid glibenclamide exists largely in the uncharged form, the instantaneous current was, in a voltage-independent manner, suppressed by the extracellular drug at micromolar concentrations without significantly affecting the depolarization-induced inactivation. At alkaline pH, at which almost all of the drug is in the charged form, glibenclamide speeded the inactivation time course and induced a leftward shift of the steady-state inactivation curve at much higher concentrations. Thus it is concluded that glibenclamide exerts inhibiting actions on swelling-activated Cl- channels from the extracellular side and that the uncharged form is mainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.