Bax and Bak are pro-apoptotic factors that are required for cell death by the mitochondrial or intrinsic pathway. Bax is found in an inactive state in the cytosol and upon activation is targeted to the mitochondrial outer membrane where it releases cytochrome c and other factors that cause caspase activation. Although Bak functions in the same way as Bax, it is constitutively localized to the mitochondrial outer membrane. In the membrane, Bak activation is inhibited by the voltage-dependent anion channel isoform 2 (VDAC2) by an unknown mechanism. Using blue native gel electrophoresis, we show that in healthy cells endogenous inactive Bak exists in a 400-kDa complex that is dependent on the presence of VDAC2. Activation of Bak is concomitant with its release from the 400-kDa complex and the formation of lower molecular weight species. Furthermore, substitution of the Bak transmembrane anchor with that of the mitochondrial outer membrane tail-anchored protein hFis1 prevents association of Bak with the VDAC2 complex and increases the sensitivity of cells to an apoptotic stimulus. Our results suggest that VDAC2 interacts with the hydrophobic tail of Bak to sequester it in an inactive state in the mitochondrial outer membrane, thereby raising the stimulation threshold necessary for permeabilization of the mitochondrial outer membrane and cell death.Apoptosis is a physiological process for eliciting cell death and is essential for the normal growth and development of multicellular organisms. Studies on gene-deleted mice have shown that the presence of either one of the pro-apoptotic Bcl-2 family members, Bak or Bax, is necessary for cell death by the "intrinsic" pathway, which can be inhibited by Bcl-2 (1, 2). In this pathway, also known as the "mitochondrial" pathway, Bak and Bax act on the mitochondrial outer membrane to cause release of molecules such as cytochrome c, which binds to Apaf-1 in the cytosol triggering formation of the apoptosome (2).The mechanisms that regulate Bak and Bax activation, and how they cause the mitochondrial outer membranes to become permeable, are not well understood. Current models for inducing cytochrome c release include the following: (a) Bak and Bax forming large pores themselves (2); (b) Bak and Bax altering the conformation of existing pores (3); (c) Bak and Bax regulating the production of long chain ceramides that may contribute to pore formation (4, 5); and (d) Bak and Bax regulating mitochondrial morphology machineries to activate MOMP 5 (6). Regardless of the mechanism, any of these possible events require Bax/ Bak activation. In contrast to Bax, which is redirected from the cytosol to mitochondria upon apoptotic induction (7), Bak is constitutively located within the mitochondrial outer membrane (8, 9). Bak has been reported to interact with a number of other proteins at the mitochondrial outer membrane, including the Bcl-2 anti-apoptotic family members Bcl-x L , Mcl-1 (10, 11), and A1 (12), the pro-apoptotic BH3-only Bcl-2 family proteins tBid, Bad, and Bim (13, 14), the mi...