The family of CLC proteins comprises both Cl-channels and Cl-/H+ exchange transporters with varying degrees of voltage-dependence.The human CLC-5 is an electrogenic voltage-dependent 2Cl-/1H+ exchanger that gives rise to strongly outwardly-rectifying currents when expressed.We conducted whole-cell recordings from HEK293 cells transiently transfected with either wild-type CLC-5 or a permeation-deficient mutant, E268A.With E268A CLC-5 we recorded transient voltage-dependent currents that represent the gating currents associated with CLC-5 activation and had kinetics that could be described by voltage-dependent forward and reverse transition rates.Extracellular solutions rich in Cl-or Br-gave a gating charge of 1.3, but this was reduced to 0.9 when replaced by the impermeant anions aspartate, methanesulfonate, sulfate, or HEPES.Extracellular ion-depletion by local perfusion with isotonic mannitol failed to reduce the gating charge further.Lowering intracellular pH from 7.4 to 5.4 did not shift the voltage-dependence of the gating currents, but reducing and increasing intracellular Cl-shifted the charge-voltage relationship to more negative and positive potentials, respectively.Our data suggest that voltage-sensing is an intrinsic property of the CLC-5 protein and that permeant anions, particularly Cl-, modulate a voltage-dependent transition to an activated state that permits ion transport.