Recently, using patch-clamp method, an ATP-sensitive potassium channel in rat liver mitochondrial inner membrane was identified. In this report we investigated the effects of different potassium channel openers on potassium ion permeability of rat liver mitochondria. For that purpose we employed light scattering technique, reflecting mitochondrial volume changes linked to solute transport. Among various potassium channel openers RP 66471, pinacidil, minoxidil sulfate and KRN 2391 were found to stimulate mitochondrial swelling with high efficiency. P1060, aprykalim and diazoxide had only a slight effect, while Ro 31-6930 and nicorandil were found ineffective. Activation by the K+ openers was abolished by the antidiabetic sulfonylurea, glibenclamide (the well known blocker of ATP-sensitive potassium channels). These findings may suggest that mitochondrial ATP-dependent K+ channel is involved in regulation of mitochondrial matrix volume.