To make biophysical measurements of functions such as the pore-forming activity of mitochondrial voltage-dependent anion-selective channel protein 1 (VDAC1), it is first necessary to obtain a source of purified VDAC protein. In this protocol, we present a method for obtaining rat liver mitochondria as a source of VDAC1 and then describe two methods, one using a nonionic detergent and the other an ionic detergent, for purifying VDAC1 from the isolated mitochondria. This produces a source of VDAC1 proteins that are suitable for subsequent incorporation into artificially prepared phospholipid bilayers. Furthermore, the isolated mitochondria can be used for assaying the mitochondrial permeability transition pore (MPTP).