CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC50 of 8.6 μM. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 ± 7.9 and 67.5 ± 6.9 picosiemens in symmetric 309 and 159 mM KCl, respectively. The anion selectivity of this activity is Br ≃ Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC50 of 86 μM at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.