We investigated the accessibility to protons and thiol-directed reagents of a cysteine substituted at position 338 in transmembrane segment 6 (TM6) of CFTR to test the hypothesis that T338 resides in the pore. Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. The pKa was shifted toward alkaline values when a nearby positive charge (R334) was substituted with neutral or negatively charged residues, consistent with the predicted influence of the positive charge of R334, and perhaps other residues, on the titration of a cysteine at 338. The relative rates of chemical modification of T338C CFTR by MTSET+ and MTSES- were also altered by the charge at 334. These observations support a model for CFTR that places T338 within the anion conduction path. The apparent pKa of a cysteine substituted at 338 and the relative rates of reaction of charged thiol-directed reagents provide a crude measure of a positive electrostatic potential that may be due to R334 and other residues near this position in the pore.