SLC12A cation/Cl− cotransporter gene family has two major branches. The K+:Cl− cotransporters (KCC1 to KCC4) that are inhibited by phosphorylation and activated by dephosphorylation, and the Na-coupled chloride cotransporters (NCC and NKCCs) that are activated by phosphorylation and inhibited by dephosphorylation. We have recently shown that the serine/threonine kinase WNK3 modulates the activity of the cation-chloride cotransporters. Here we show that co-injection of Xenopus laevis oocytes with any of the K+:Cl− cotransporters cRNA, together with the wild-type WNK3 cRNA results in prevention of the cell swelling induced activation of the cotransporters (see 86Rb+ uptake per group in nmol/oocyte/h in the table). In contrast, co-injection of the K+:Cl− cotransporters cRNA with the catalytically inactive WNK3-D294A, resulted in a markedly increase of the cotransporters activity, even in isotonic conditions in which the K+:Cl− cotransporters are normally inactive (see table). The activation of KCC1 to KCC4 by WNK3-D294A in isotonicity can be completely prevented by treatment of oocytes with a combination of calyculin A and cyclosporine A, which inhibit the protein phosphatase 1 and 2B, respectively. Thus, the catalytically inactive WNK3-D294A bypasses the tonicity requirements for K+:Cl− cotransporters activation via a phosphatases dependent mechanism.