5-Lipoxygenase (5-LO) initiates the biosynthesis of proinflammatory leukotrienes from arachidonic acid (AA). Here, we demonstrate that hypertonicity suppresses ionophore-induced 5-LO product formation reversibly in isolated human polymorphonuclear leukocytes (PMNL) and in Mono Mac 6 cells. Hypertonicity blocked the liberation of AA and abrogated translocation of 5-LO to the nuclear membrane. Accordingly, in the presence of exogenous AA, 5-LO product formation was less affected. The effects of hypertonicity were a result of cell shrinkage and not cytosolic hyperosmolarity. Hypertonicity did not inhibit the rapid increase in intracellular Ca(2)(+) induced by ionophores but prevented the ionophore-induced activation of p38 MAPK-regulated MAPKAP kinases, which can phosphorylate and activate 5-LO (and cPLA(2)). In summary, we show that hypertonicity blocks agonist-induced release of AA, 5-LO product formation, and translocation and in parallel, prevents activation of p38 MAPK and downstream 5-LO kinases in leukocytes.