Apoptosis plays a critical role in many neurologic diseases, including stroke. Cytochrome c release and activation of various caspases are known to occur after focal and global ischemia. However, recent reports indicate that caspase-independent pathways may also be involved in ischemic damage. Apoptosis-inducing factor (AIF) is a novel flavoprotein that helps mediate caspase-independent apoptotic cell death. AIF translocates from mitochondria to nuclei where it induces caspase-independent DNA fragmentation. Bcl-2, a mitochondrial membrane protein, protects against apoptotic and necrotic death induced by different insults, including cerebral ischemia. In the present study, Western blots confirmed that AIF was normally confined to mitochondria but translocated to nuclei or cytosol 8, 24, and 48 hours after onset of ischemia. Overall, AIF protein levels also increased after stroke. Confocal microscopy further demonstrated that nuclear AIF translocation occurred in the peri-infarct region but not in the ischemic core where only some cytosolic AIF release was observed. Our data also suggest that AIF translocated into nuclei after cytochrome c was released into the cytosol. Bcl-2 transfection in the peri-infarct region blocked nuclear AIF translocation and improved cortical neuron survival.