Transient forebrain or global ischemia induces delayed neuronal death in vulnerable CA1 pyramidal cells with many features of apoptosis. A brief period of ischemia, i.e., ischemic preconditioning, affords robust protection of CA1 neurons against a subsequent more prolonged ischemic challenge. Here we show that preconditioning acts via PI3K/Akt signaling to block the ischemia-induced cascade involving mitochondrial translocation of Bad, assembly of Bad with Bcl-x(L), cleavage of Bcl-x(L) to form its prodeath fragment, DeltaN-Bcl-x(L), activation of large-conductance channels in the mitochondrial outer membrane, mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), caspase activation, and neuronal death. These findings show how preconditioning acts to prevent the release of cytochrome c and Smac/DIABLO from mitochondria and to preserve the integrity of the mitochondrial membrane. The specific PI3K inhibitor LY294002 administered in vivo 1 h before or immediately after ischemia or up to 120 h later significantly reverses preconditioning-induced protection, indicating a requirement for sustained PI3K signaling in ischemic tolerance. These findings implicate PI3K/Akt signaling in maintenance of the integrity of the mitochondrial outer membrane.