The Bcl-2 family of proteins is crucial for apoptosis regulation. Members of this family insert through a specific C-terminal anchoring transmembrane domain (TMD) in the mitochondrial outer membrane where they hierarchically interact to determine cell fate. While the mitochondrial membrane has been proposed to actively participate in these protein-protein interactions, the influence of the TMD in the membrane-mediated interaction is poorly understood. Synthetic peptides (TMD-pepts) corresponding to the putative TMD of anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w and Mcl-1) and pro-apoptotic (Bax, Bak) members were synthesized and characterized. TMD-pepts bound more efficiently to mitochondria-like bilayers than to plasma membrane-like bilayers, and higher binding correlated with greater membrane perturbation. The Bcl-2 TMD peptides promoted MOM permeabilization and cytochrome c release from isolated mitochondria and different cell lines. TMD-pepts exhibited non-apoptotic pro-death activity when apoptosis stimuli were absent. In addition, the peptides enhanced the apoptotic pathway induced by chemotherapeutic agents in co-treatment. These results clearly demonstrate that biophysical and molecular analysis of these membrane interactions reveal new insights into the participation of the mitochondrial membrane in Bcl-2 family protein-protein interactions. Moreover, the membrane perturbation effects of the TMD-pepts observed in this study open the way to their use as new chemical tools to sensitize tumor cells to chemotherapeutic agents, in accordance with the concept of mitochondria priming.