It has been shown that the voltage (Vm) dependence of ClC Cl− channels is conferred by interaction of the protopore gate with H+ ions. However, in this paper we present evidence which indicates that permeant Cl− ions contribute to Vm-dependent gating of the broadly distributed ClC-2 Cl− channel. The apparent open probability (PA) of ClC-2 was enhanced either by changing the [Cl−]i from 10 to 200 mm or by keeping the [Cl−]i low (10 mm) and then raising [Cl−]o from 10 to 140 mm. Additionally, these changes in [Cl−] slowed down channel closing at positive Vm suggesting that high [Cl−] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the PA(Vm) curves were nearly identical with Cl− or Br− but shifted to negative voltages in the presence of SCN− ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN−/Cl− mixture in agreement with the idea that pore occupancy by different permeant anions modifies the Vm dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl− ions dwell in the central binding site. In contrast, when Cl− ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl−-dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl−]o enhances channel opening when the [Cl−]i is in the physiological range.