In Ca²⁺ionophore-activated HL-60 granulocytes the mitogen-activated protein kinase kinase-1 inhibitor, PD098059, blocked translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and the corresponding enzyme activation. PD098059 inhibited 5-HETE formation with an IC₅₀= 9.4 μMin cells stimulated with A23187 alone, and with an IC₅₀= 12 μMin cells stimulated with A23187 plus 20 μMarachidonic acid. PD098059 inhibited translocation of 5-lipoxygenase in a concentration-dependent manner with an IC₅₀approximately 10 μM. At concentrations less than 100 μMPD098059 had no effect on purified recombinant 5-LO activity. Collectively, these data indicate that MAPKK-1 participates in the molecular processes governing activation and translocation of 5-lipoxygenase from the cytosol to the nuclear membrane.