5-Lipoxygenase (5-LO) is a key enzyme involved in the synthesis of leukotrienes from arachidonic acid, and its activation is usually followed by translocation to the nuclear envelope. The details of mechanisms involved in the translocation of 5-LO are not well understood, though Ca2+ is known to be essential. Here we show that ionomycin, a Ca2+ ionophore, induces 5-LO translocation and necrotic cell death in Rat-2 fibroblasts, suggesting a potential relationship between activation of 5-LO and cell death. These effects were markedly attenuated in Rat2-Rac(N17) cells expressing a dominant negative Rad mutant. Pretreatment with SB203580, a specific inhibitor of p38 MAP kinase, or EGTA, a Ca2+ chelator, likewise diminished ionomycin-induced 5-LO translocation and cell death, but PD98059, a MEK inhibitor, did not. Thus, Rac and p38 MAP kinase appear to be components in a Ca2+ dependent pathway leading to 5-LO translocation and necrotic cell death in Rat-2 fibroblasts.