Short-term regulation of members of the Na-K-Cl cotransporter family takes place by phosphorylation/dephosphorylation events. Three NH(2)-terminal threonines have been previously identified as phosphoacceptors involved in activation of the ubiquitous/secretory Na-K-Cl cotransporter (NKCC1). In this study, we demonstrate that the corresponding threonines are also involved in the regulation of the renal Na-K-Cl cotransporter (NKCC2). The transport activity of NKCC2, exogenously expressed in Xenopus laevis oocytes, is shown to be stimulated by hypertonicity. Mutagenic analysis demonstrated that threonines T99, T104, and T117 comprise a regulatory domain responsible for the activation of NKCC2 in hypertonic solutions: although none of the threonines was found to be individually necessary or sufficient for regulation, the three residues together are required to obtain the full hypertonic response. Under isotonic and hypotonic conditions, NKCC2 retains 50% of its activity in the absence of phosphorylation of the threonine-regulatory domain. Selective deletions of peptide segments revealed only a minor role for the NH(2)-terminal cytosolic domain of NKCC2 upstream of the threonine regulatory domain, including the recently identified proline alanine-rich Ste-20-related kinase-binding motif. A chimeric NKCC containing the first 104 amino acids of NKCC1 on the NKCC2 backbone behaved essentially the same as NKCC2, further arguing against a major role for this upstream region in NKCC2 regulation.