Programmed cell death is essential in organ development and tissue homeostasis and its deregulation is associated with the development of several diseases in mice and humans. The precise mechanisms that control cell death have not been elucidated fully, but it is well established that this form of cellular demise is regulated by a genetic program which is activated in the dying cell. Here we report the identification, cloning and characterization of harakiri, a novel gene that regulates apoptosis. The product of harakiri, Hrk, physically interacts with the death-repressor proteins Bcl-2 and Bcl-X(L), but not with death-promoting homologs, Bax or Bak. Hrk lacks conserved BH1 and BH2 regions and significant homology to Bcl-2 family members or any other protein, except for a stretch of eight amino acids that exhibits high homology with BH3 regions. Expression of Hrk induces cell death which is inhibited by Bcl-2 and Bcl-X(L). Deletion of 16 amino acids including the conserved BH3 region abolished the ability of Hrk to interact with Bcl-2 and Bcl-X(L) in mammalian cells. Moreover, the killing activity of this mutant form of Hrk (Hrk deltaBH3) was eliminated or dramatically reduced, suggesting that Hrk activates cell death at least in part by interacting with and inhibiting the protection afforded by Bcl-2 and Bcl-X(L). Because Hrk lacks conserved BH1 and BH2 domains that define Bcl-2 family members, we propose that Hrk and Bik/Nbk, another BH3-containing protein that activates apoptosis, represent a novel class of proteins that regulate apoptosis by interacting selectively with survival-promoting Bcl-2 and Bcl-X(L).