Enhanced formation of reactive oxygen species (ROS), superoxide (O 2 ·−), and hydrogen peroxide (H 2O 2) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O 2 ·− elicited rapid and massive cytochrome c release (CCR), whereas H 2O 2 failed to induce any release. Both O 2 ·− and H 2O 2 promoted activation of the mitochondrial permeability transition pore by Ca 2+, but Ca 2+-dependent pore opening was not required for O 2 ·−-induced CCR. Furthermore, O 2 ·− alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O 2 ·−-induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O 2 ·− exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O 2 ·−-induced CCR did not depend on Bax translocation to mitochondria. O 2 ·−-induced CCR was followed by caspase activation and execution of apoptosis. Thus, O 2 ·− triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.