Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl(-) transport. Reductions in either cell volume or cytosolic Cl(-) concentration ([Cl(-)](i)) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl(-)](i) was evaluated in model duck red blood cells. Replacement of [Cl(-)](i) with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl(-)](i) at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl(-)](i) is associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl(-)](i) accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl(-)] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl(-)]. The major effect of Cl(-) on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein.