Two important questions on the molecular mechanism of the B cell CLL/lymphoma 2 (BCL2) proteins involve the interaction network between pro-and antiapoptotic members and the role of their translocation to the mitochondrial membrane during apoptosis. We used fluorescence correlation spectroscopy to quantify the molecular interactions of BH3-interacting domain death agonist (BID) and its truncated form tBID with the B cell lymphoma extra-large protein truncated at the C terminus (BCL XL ΔCt) in solution and in membranes, and we found that (i) only the active form tBID binds to BCL XL ΔCt and (ii) that the membrane strongly promotes binding between them. Particularly, a BH3 peptide from BID disrupts the tBID-BCL XL complex in solution, but only partially in lipid bilayers. These data indicate that tBID-BCL XL interactions in solution and lipid membranes are distinct, and they support a model in which BCL XL inhibition of tBID takes place predominantly at the membrane. Our findings imply an active role of the membrane in modulating the interactions between BCL2 proteins that has so far been underestimated.