The phospholipid cardiolipin is thought to play an important role in mitochondrial physiology and mitochondrial outer membrane permeabilization (MOMP) during apoptosis. However, this remains controversial due to the lack of good cellular models. To address this issue, we generated a human cell line completely depleted of its cardiolipin content. Cardiolipin-deficient cells were viable, but growth was dramatically affected when cells were forced to use oxidative phosphorylation as an energy source, pointing to a defective respiratory chain. When we looked at MOMP and apoptosis in these cells, we found that they proceeded normally in the absence of cardiolipin. We also downregulated the mitochondrial protein MTCH2, a putative receptor for tBID, in wild type and cardiolipin-deficient cells. In apoptotic conditions, we observed a strong decrease in tBID recruitment when both cardiolipin and MTCH2 were depleted, suggesting that cardiolipin and MTCH2 might play a redundant function in the recruitment of tBID to mitochondria.