Mutations within the with-no-K(Lys) (WNK) kinases cause Gordon's syndrome characterized by hypertension and hyperkalaemia. WNK kinases phosphorylate and activate the STE20/SPS1-related proline/alanine-rich kinase (SPAK) protein kinase, which phosphorylates and stimulates the key Na(+):Cl(-) cotransporter (NCC) and Na(+):K(+):2Cl(-) cotransporters (NKCC2) cotransporters that control salt reabsorption in the kidney. To define the importance of this pathway in regulating blood pressure, we generated knock-in mice in which SPAK cannot be activated by WNKs. The SPAK knock-in animals are viable, but display significantly reduced blood pressure that was salt-dependent. These animals also have markedly reduced phosphorylation of NCC and NKCC2 cotransporters at the residues phosphorylated by SPAK. This was also accompanied by a reduction in the expression of NCC and NKCC2 protein without changes in messenger RNA (mRNA) levels. On a normal Na(+)-diet, the SPAK knock-in mice were normokalaemic, but developed mild hypokalaemia when the renin-angiotensin system was activated by a low Na(+)-diet. These observations establish that SPAK plays an important role in controlling blood pressure in mammals. Our results imply that SPAK inhibitors would be effective at reducing blood pressure by lowering phosphorylation as well as expression of NCC and NKCC2. See accompanying Closeup by Maria Castañeda-Bueno and Gerald Gamba (DOI 10.1002/emmm.200900059).