The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.