Abstract The intrinsic apoptosis signaling pathway comprises the intimate interaction network of the Bcl-2 protein family, in which both pro- and antiapoptotic members pushing cell fate to either survival or death. As a critical event, oligomerization of pore-forming effector proteins BAX, BAK and BOK drive mitochondrial outer membrane permeabilization (MOMP), which results in Cytochrome C release and ultimately apoptotic cell death. Thus far, investigations focus on protein-protein interactions mediated by the BH3-domain and hydrophobic groove. These interactions are targeted by BH3 mimetics that represent highly efficient anti-cancer drugs. In contrast, the C-terminal α-9 helices, known as transmembrane domains (TMD), tend to be neglected as a potential secondary interaction site with other Bcl-2 proteins. However, TMDs strongly affect subcellular localization and there is increasing evidence that TMDs crucially influence Bcl-2 protein interactions and therefore apoptosis signaling. Although TMDs are widely abundant in pro- and anti-apoptotic Bcl-2 proteins, their contribution to the complex Bcl-2 interaction network remains largely in the dark. Aiming to shed more light on the interactions of TMD among effector Bcl-2 proteins, we generated a bimolecular complementation assay in which effector TMD peptides are coupled to a bimolecular split-luciferase system. In addition, vectors encode for simultaneous expression of fluorophores to allow control of transgene expression. Human cell lines were co-transfected with combinations of TMD expression constructs yielding luminescence-based interaction data with a fluorescence-based normalization. Interestingly, we found an almost exclusively homotypic TMD interaction among the TMDs of BAX, BAK and BOK. Moreover, confocal microscopy with modified fluorophore-coupled TMD peptides revealed that TMD sequences were sufficient to locate BAX TMD and BAK TMD peptides effectively to mitochondria, while BOK TMD peptides were mainly localized to the endoplasmic reticulum. Strikingly, exchanging TMD sequences in full-length effector proteins profoundly modulated cell death induction. These results strongly suggest a central functional relevance of the α-9-helices in promoting homotypic interactions of effectors BAX, BAK and BOK during oligomerization and pore formation. In contrast, putative heterotypic interaction among effector proteins is not reflected by the newly generated TMD interaction assay. Thus, effector TMDs are a vital factor for specific protein localization and cell death induction displaying specific functionality in each effector protein. Our findings underline the importance of further investigating the TMD region as a potential oncogenic feature influencing interaction of Bcl-2 proteins and therefore as a potential target for therapeutic intervention. Citation Format: Tobias B. Beigl, Sandra Weller, Benjamin Schäfer, Kathrin Böpple, Lara-Sophie Rieder, Hans-Georg Kopp, Markus Rehm, Walter E. Aulitzky, Frank Essmann. Brought to light: the homotypic interaction pattern of Bcl-2 effector transmembrane domains in a bimolecular luciferase complementation assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1947.