Although there have been a number of recent studies on the role of Rac in the generation of reactive oxygen species (ROS), details of the signalling pathway remain unclear. In the present study we analysed the extent to which the activation of cytosolic phospholipase A(2) and the resultant release of arachidonic acid (AA) are involved in the Rac-mediated generation of ROS. Transfection of Rat-2 cells with RacV12, a constitutively active form of Rac1, induced elevated levels of ROS, as reflected by increased H(2)O(2)-sensitive fluorescence of 2', 7'-dichlorofluorescein. These effects could be blocked by inhibiting phospholipase A(2) or 5-lipoxygenase but not by inhibiting cyclo-oxygenase. The application of exogenous AA increased levels of ROS but the effect was dependent on the further metabolism of AA to leukotrienes C(4)/D(4)/E(4) by 5-lipoxygenase. Indeed, the exogenous application of a mixture of leukotrienes C(4)/D(4)/E(4) elicited transient elevations in the levels of ROS that were blocked by catalase. These findings indicate that phospholipase A(2) and subsequent AA metabolism by 5-lipoxygenase act as downstream mediators in a Rac signalling pathway leading to the generation of ROS.