Cell spreading and migration are regulated in a Rho family GTPase-dependent manner by growth factors and integrin-mediated cell-extracellular matrix (ECM) interactions. The molecular mechanisms involved in the ECM- and growth factor-mediated activation of these small GTPases remain unclear. In the present study, we demonstrate that integrin-linked kinase (ILK), which is a focal adhesion protein activated by both ECM and growth factors, is required for the activation of Rac and Cdc42 in epithelial cells. Ectopic expression of active ILK in mammary epithelial cells induces dramatic reorganization of the actin cytoskeleton and promotes rapid cell spreading on fibronectin. These effects are associated with constitutive activation of both Rac and Cdc42, but not Rho. The use of ILK siRNA or small molecule inhibitors to inhibit ILK expression and kinase activity, respectively, results in diminished cell spreading and actin cytoskeleton reorganization, concomitant with a reduction in Rac and Cdc42 activation. Studies into the mechanism of ILK-mediated Rac activation suggest an important role for the ILK-beta-parvin interaction and the activity of the Rac/Cdc42-specific guanine nucleotide exchange factor alpha-PIX downstream of ILK. Taken together, these data demonstrate an essential role of ILK kinase activity in Rac- and Cdc42-mediated actin cytoskeleton reorganization in epithelial cells, further solidifying a role for ILK in the regulation of cancer cell motility and invasiveness.