Previous studies have identified several transmembrane segments (TMs), including TM1, TM3, TM6, TM9, TM11, and TM12, as pore-lining segments in cystic fibrosis transmembrane conductance regulator (CFTR), but the role of TM5 in pore construction remains controversial. In this study, we employed substituted cysteine accessibility methodology (SCAM) to screen the entire TM5 defined by the original topology model and its cytoplasmic extension in a Cysless background. We found six positions (A299, R303, N306, S307, F310, and F311) where engineered cysteines react to intracellular 2-sulfonatoethyl methanethiosulfonate (MTSES-). Quantification of the modification rate of engineered cysteines in the presence or absence of ATP suggests that these six residues are accessible in both the open and closed states. Whole-cell experiments with external MTSES- identified only two positive positions (L323 and A326), resulting in a segment containing 11 consecutive amino acids, where substituted cysteines respond to neither internal nor external MTSES-, a unique feature not seen previously in CFTR's pore-lining segments. The observation that these positions are inaccessible to channel-permeant thiol-specific reagent [Au(CN)2]2' suggests that this segment of TM5 between F311 and L323 is concealed from the pore by other TMs and/or lipid bilayers. In addition, our data support the idea that the positively charged arginine at position 303 poses a pure electrostatic action in determining the single-channel current amplitude of CFTR and the effect of an open-channel blocker glibencalmide. Collectively, we conclude that the cytoplasmic portion of CFTR's TM5 lines the pore. Our functional data are remarkably consistent with predicted structural arrangements of TM5 in some homology models of CFTR.