Cleaved or truncated BID (tBID) is known to oligomerize both BAK and BAX. Previously, BAK and BAX lacing the C-terminal fragment (BAXΔC) were shown to induce modest cytochrome c (Cyt c) release from rat brain mitochondria when activated by tBID. We now show that tBID plus monomeric full-length BAX induce extensive release of Cyt c, Smac/DIABLO, and Omi/HtrA2 (but not endonuclease G and the apoptosis inducing factor) comparable to the release induced by alamethicin. This occurs independently of the permeability transition without overt changes in mitochondrial morphology. The mechanism of the release may involve formation of reactive oxygen species (ROS) and activation of calcium-independent phospholipase A2 (iPLA2). Indeed, increased ROS production and activated iPLA2 were observed prior to massive Cyt c release. Furthermore, the extent of inhibition of Cyt c release correlated with the degree of suppression of iPLA2 by the inhibitors propranolol, dibucaine, 4-bromophenacyl bromide, and bromenol lactone. Consistent with a requirement for iPLA2 in Cyt c release from brain mitochondria, synthetic liposomes composed of lipids mimicking the outer mitochondrial membrane (OMM) but lacing iPLA2 failed to release 10 kDa fluorescent dextran (FD-10) in response to tBID plus BAX. We propose that tBID plus BAX activate ROS generation, which subsequently augments iPLA2 activity leading to changes in the OMM that allow translocation of certain mitochondrial proteins from the intermembrane space.