The outer membrane of rat liver mitochondria contains a channel-forming protein known as VDAC (voltage-dependent anion-selective channel). This protein has been functionally purified by a combination of ion exchange chromatography, gel filtration and affinity chromatography on a Concanavalin A-containing column. An estimated 300-fold purification was achieved over the specific activity in mitochondrial membranes. When the purified protein is run on an SDS polyacrylamide gel, essentially only one band is present at a position consistent with a molecular weight of 32,000. The resulting protein is functional and behaves normally based on channel size, selectivity and voltage dependence.