The Bcl-2-sensitive release of proteins such as cytochrome c from the mitochondrial intermembrane space into the cytosol is a critical early event in apoptosis. The mitochondrial permeability transition is also an important event in many forms of apoptotic cell death. To determine whether the permeability transition led to the release of apoptogenic proteins from mitochondria we induced the permeability transition in isolated rat liver mitochondria and characterised the proteins which were released. The permeability transition led to a generalised, non-specific release of proteins, including cytochrome c, from the mitochondrial intermembrane space which was prevented by an inhibitor of the permeability transition. To determine the mechanism of this protein release we measured both mitochondrial matrix swelling and protein release during the permeability transition in media of different osmolarities. Protein release correlated with mitochondrial matrix swelling, therefore the permeability transition causes release of proteins from the intermembrane space by rupturing the mitochondrial outer membrane. Supporting an apoptotic role for the proteins released by this mechanism, supernatants from mitochondria undergoing the permeability transition caused apoptotic changes in isolated nuclei. These data support the proposal that the mitochondrial permeability transition can induce apoptosis by releasing apoptogenic proteins into the cytoplasm [Skulachev, V.P., FEBS Lett. 397 (1996) 7–10].