Abstract Effect of potassium channel openers on membrane potential of rat liver mitochondria was studied. It has been found that potassium channel opener RP 65471 induces depolarization of the mitochondrial membrane. Since neither the inhibition of mitochondrial respiration nor the uncoupling of mitochondria was observed concomitantly, the specific effect on the mitochondrial potential is postulated. Most likely the effect is caused by the increase of permeability of the inner mitochondrial membrane to potassium ions. Interestingly, however, it was found that no other potassium channel openers tested but RP 66471 was able to induce depolarization of mitochondrial membrane.