A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current (i)-voltage (V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current (I)-V relationship (I = N × P_o × i, where N is the number of active channels and P_o is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the P_o of V317E CFTR is voltage dependent. The decrease in P_o at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P_o-V relationship did not depend on the direction of Cl movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.