CFTR is the only ABC transporter functioning as a chloride (Cl−) channel.We studied molecular determinants, which might distinguish CFTR from standard ABC transporters, and focused on the interface formed by the intracellular loops from the membrane spanning domains. Methods: Residues from ICL2 and ICL4 in close proximity were targeted, and their involvement in the functioning of CFTR was studied by whole cell patch clamp recording. Results: We identified 2 pairs of amino acids, at the extremity of the bundle formed by the four intracellular loops, whose mutation i) decreases the Cl− current of CFTR (couple E267-K1060) or ii) increases it with a change of the electrophysiological signature (couple S263-V1056). Conclusions: These results highlight the critical role of these ICL residues in the assembly of the different domains and/or in the Cl− permeation pathway of CFTR.