Cell shrinkage phosphorylates and activates the Na-K-2Cl cotransporter (NKCC1), indicating the presence of a volume-sensitive protein kinase. To identify this kinase, extracts of normal and shrunken aortic endothelial cells were screened for phosphorylation of NKCC1 fusion proteins in an in-the-gel kinase assay. Hypertonic shrinkage activated a 46-kDa kinase that phosphorylated an NH2-terminal fusion protein, with weaker phosphorylation of a COOH-terminal fusion protein. This cytosolic kinase was activated by both hypertonic and isosmotic shrinkage, indicating regulation by cell volume rather than osmolarity. Subsequent studies identified this kinase as c-Jun NH2-terminal kinase (JNK). Immunoblotting revealed increased JNK activity in shrunken cells; there was volume-sensitive phosphorylation of NH2-terminal c-Jun fusion protein; immunoprecipitation of JNK from shrunken cells but not normal cells phosphorylated NKCC1 in gel kinase assays; and treatment of cells with tumor necrosis factor, a known activator of JNK, mimicked the effect of hypertonicity. We conclude that JNK is a volume-sensitive kinase in endothelial cells that phosphorylates NKCC1 in vitro. This is the first demonstration of a volume-sensitive protein kinase capable of phosphorylating a volume-regulatory transporter.