Cl- and HCO3- are the two major cellular anions. Surprisingly, a role of intracellular Cl- (Cl-in) as a signaling molecule that regulates the activity of other transporters has not been evaluated. We found that Cl-in functions as a regulator of cellular Na+ and HCO3- concentrations by regulating the activity of several NBCs. All forms of regulation by Cl-in are mediated by the Cl- interacting GXXXP motifs. In the basal state NBCe1-B is inhibited by high Cl-in, while NBCe1-A is resistant to Cl-in between 5-140 mM. By contracts, the IRBIT-activated NBCe1-B and the basal activity of NBCe2-C are inhibited by high affinity Cl-in sites, with apparent affinity of about 10 mM. The NBCe1-B high and low affinity Cl-in sites are mediated by separate GXXXP motifs. Mutations in the GXXXP motifs in the autoinhibitory site of NBCe1-B and the GXXXP motif in the N terminus of NBCe2-C eliminated inhibition by low Cl-in, while sparing inhibition of NBCe1-B by high Cl-in. Mutation of a second N terminus GXXXP motif was required to eliminate inhibition of NBCe1-B by Cl-in. Deletion of residues 29-41 of NBCe1-A uncovered inhibition by Cl-in that was mediated by a hidden GXXXP motif homologous with the NBCe1-B low affinity site. These finding reveal a novel Cl-in sensing mechanism that is transmitted by regulation of Na+ and HCO3- transporters with major implication for cellular Na+ and HCO3- homeostasis and epithelial fluid and electrolyte secretion.