The study describes the identification and characterization of apoptosis-inducing factor (AIF), a mitochondrial protein that induces apoptosis. AIF is a flavoprotein with a molecular mass of 57,000, which shares homology with bacterial oxidoreductases. It is normally confined to mitochondria but translocates to the nucleus during apoptosis. Recombinant AIF causes chromatin condensation and DNA fragmentation in isolated nuclei and induces the release of cytochrome c and caspase-9 from mitochondria. Microinjection of AIF into cells leads to chromatin condensation, mitochondrial transmembrane potential dissipation, and phosphatidylserine exposure in the plasma membrane. These effects are not blocked by the caspase inhibitor Z-VAD.fmk. Overexpression of Bcl-2, which controls mitochondrial permeability transition pore opening, prevents AIF release from mitochondria but does not affect its apoptotic activity. These findings indicate that AIF is a mitochondrial effector of apoptotic cell death. The opening of the mitochondrial permeability transition pore, controlled by Bcl-2 family members, is a critical event in apoptosis, leading to increased outer mitochondrial membrane permeability and the release of soluble proteins. The mitochondrial intermembrane space contains several potentially apoptotic factors, including cytochrome c, procaspases 2, 3, and 9, and AIF, which induces isolated nuclei to adopt an apoptotic morphology. A purified AIF activity was maintained in the presence of the caspase inhibitor. The amino acid sequences of mouse and human AIF were aligned with those of the bacterial enzyme BDSF, showing 30% identity. The sequence includes mitochondrial localization sequences (MLS) and putative nuclear-localization sequences (NLS). The underlined sequence in mouse AIF matches mass spectroscopy data obtained from purified AIF. The GenBank accession numbers for mouse and human AIF are AF100927 and AF100928, respectively.The study describes the identification and characterization of apoptosis-inducing factor (AIF), a mitochondrial protein that induces apoptosis. AIF is a flavoprotein with a molecular mass of 57,000, which shares homology with bacterial oxidoreductases. It is normally confined to mitochondria but translocates to the nucleus during apoptosis. Recombinant AIF causes chromatin condensation and DNA fragmentation in isolated nuclei and induces the release of cytochrome c and caspase-9 from mitochondria. Microinjection of AIF into cells leads to chromatin condensation, mitochondrial transmembrane potential dissipation, and phosphatidylserine exposure in the plasma membrane. These effects are not blocked by the caspase inhibitor Z-VAD.fmk. Overexpression of Bcl-2, which controls mitochondrial permeability transition pore opening, prevents AIF release from mitochondria but does not affect its apoptotic activity. These findings indicate that AIF is a mitochondrial effector of apoptotic cell death. The opening of the mitochondrial permeability transition pore, controlled by Bcl-2 family members, is a critical event in apoptosis, leading to increased outer mitochondrial membrane permeability and the release of soluble proteins. The mitochondrial intermembrane space contains several potentially apoptotic factors, including cytochrome c, procaspases 2, 3, and 9, and AIF, which induces isolated nuclei to adopt an apoptotic morphology. A purified AIF activity was maintained in the presence of the caspase inhibitor. The amino acid sequences of mouse and human AIF were aligned with those of the bacterial enzyme BDSF, showing 30% identity. The sequence includes mitochondrial localization sequences (MLS) and putative nuclear-localization sequences (NLS). The underlined sequence in mouse AIF matches mass spectroscopy data obtained from purified AIF. The GenBank accession numbers for mouse and human AIF are AF100927 and AF100928, respectively.