Bax is a Bcl-2 family protein with pro-apoptotic activity that can form ion channels in artificial lipid membranes. The localization of Bax has been shown to change from the cytosol to the mitochondria during apoptosis. The protein can trigger cytochrome c release from mitochondria both in vitro and in vivo. In cytosol of untreated HeLa cells and in cytosolic extract from mouse liver Bax is present as a monomer with an apparent molecular weight of 24,000 Da. In mitochondria from untreated HeLa cells Bax is found as a monomer which can be removed by sodium carbonate treatment, showing that the protein is not integrated in the mitochondrial membrane. After treatment of HeLa cells with the apoptosis inducer staurosporine the monomeric cytosolic Bax concentration decreases. In the staurosporine treated cells Bax associated with the mitochondria is present as both a monomer and a large molecular weight complex. The monomer is removed from the mitochondria by sodium carbonate treatment whereas the complex is resistant to the treatment, indicating that the Bax complex is integrated into the mitochondrial membrane. The outer mitochondrial membrane protein VDAC is not co-migrating with the large molecular weight Bax complex. These results suggest that monomeric Bax is inactive in the mitochondria and Bax oligomerization or complex formation with an unknown protein is required for activity.