The cystic fibrosis transmembrane conductance regulator (CFTR) is both a member of the ATP-binding cassette superfamily and a Cl--selective ion channel. We investigated the permeation pathway of human CFTR with measurements on conduction and open-channel blockade by diphenylamine-2-carboxylic acid (DPC). We used site-directed mutagenesis and oocyte expression to locate residues in transmembrane domain (TM) 6 and TM 12 that contact DPC and control rectification and singlechannel conductances. Thus, TM 12 and the previously investigated TM 6 line the CFTR pore. In each TM, residues in contact with DPC are separated by two turns of an a helix. The contributions of TM 6 and TM 12 to DPC block and Cl-permeation, however, are not equivalent. The resulting structural model for the conduction pathway may guide future studies of permeation in other Cl-channels and ATP-binding cassette transporters.