Tumor necrosis factor alpha (TNF alpha) activates various signal transduction pathways including those involving phosphatidylinositol. 3-kinase (PI3K), extracellular signal-regulated kinases (Erk), c-Jun N-terminal protein kinases (JNK), and p38 kinases. Using the Rac binding domain of PAK (PAK-RBD) as an activation-specific probe, here we demonstrate that TNF alpha very rapidly and transiently activates the Rho family GTPase Rac in L929 cells. The PI3K inhibitor LY294002 significantly inhibited TNF alpha activation of Rac as well as Erk and abolished that of the PI3K target Akt, without showing any inhibitory effects on JNK and p38 activation. Furthermore, TNF alpha activation of Erk was abolished by a dominant negative Rac mutant, Rac17N, or by an activated Rac mutant, Rac12V. These findings suggest that Rac is activated by a mechanism that is at least partly dependent on PI3K in TNF alpha stimulated cells and plays a critical role in activation of the Erk signaling pathway.