A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl- as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3-, BrO3-); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl- at the regulatory binding site but impair Cl- passage through the channel pore (Br-, NO3-, ClO3-, I-, ClO4-, SCN-). The permeability sequence for rClC-1, SCN- approximately ClO4- > Cl- > Br- > NO3- approximately ClO3- > I- >> BrO3- > HCO3- >> methanesulfonate approximately cyclamate approximately glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN- approximately ClO4- > I- > NO3- approximately ClO3- approximately methanesulfonate > Br- > cyclamate > BrO3- > HCO3- > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be approximately 4.5 A. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl- and SCN- or ClO4- suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.