The purpose of the present study was to investigate the effect exerted by the antidiabetic sulfonylurea, glibenclamide, a well known blocker of ATP-regulated potassium channels, on membrane potential and proton gradient in rat liver mitochondria. Membrane potential of mitochondria was determined using a TPP+ selective electrode. Mitochondrial proton gradient value was measured with 2',7',bis(carboxyethyl)-5,6-carboxyfluorescein, the pH-sensitive fluorescent probe. Mitochondrial membrane potential and proton gradient were found to be dissipated by glibenclamide in a concentration-dependent manner, with IC50 of 70 +/- 7 microM. The present results provide evidence that the antidiabetic sulfonylurea, glibenclamide, uncouples rat liver mitochondria.