Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unknown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.