The mechanism by which the proapoptotic Bcl-2 family members Bax and Bak release cytochrome c from mitochondria is incompletely understood. In this paper, we show that activator BH3-only proteins bind tightly but transiently to the Bak hydrophobic BH3-binding groove to induce Bak oligomerization, liposome permeabilization, mitochondrial cytochrome c release, and cell death. Analysis by surface plasmon resonance indicated that the initial binding of BH3-only proteins to Bak occurred with similar kinetics with or without detergent or mitochondrial lipids, but these reagents increase the strength of the Bak-BH3-only protein interaction. Point mutations in Bak and reciprocal mutations in the BH3-only proteins not only confirmed the identity of the interacting residues at the Bak-BH3-only protein interface but also demonstrated specificity of complex formation in vitro and in a cellular context. These observations indicate that transient protein-protein interactions involving the Bak BH3-binding groove initiate Bak oligomerization and activation.