The Bcl-2 family of proteins is crucial for apoptosis regulation. The Bcl-2 proteins are synthesized in cytosolic ribosomes before reaching the lipid bilayer, where they insert through a specific C-terminal anchoring transmembrane domain. The members of this family of proteins target distinct intracellular membranes, including the mitochondrial outer membrane where they interact in order to determine cell fate. While the mitochondrial membrane has been proposed to actively participate in these protein-protein interactions, the influence of the transmembrane domain in the membrane-mediated interactions is poorly understood. The putative transmembrane domain (TMD) of anti-apoptotic (Bcl-2, Bcl-xL, Bcl-w and Mcl-1) and pro-apoptotic (Bax, Bak) members were selected and synthetic peptides (TMDpepts) derived from the respective domains were synthesized and characterized. TMDpepts showed higher affinity to interact with mitochondria-like than with plasma membrane-like model bilayers and higher binding correlated with greater membrane perturbation. These TMDpepts were effective as tools to prepare mitochondria for apoptosis by sensitizing human cervix adenocarcinoma cells to chemotherapeutic agents.