Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.