Sprouty (SPRY) protein negatively modulates fibroblast growth factor and epidermal growth factor actions. We showed that human SPRY2 inhibits cell growth and migration in response to serum and several growth factors. Using rat intestinal epithelial (IEC-6) cells, we investigated the involvement of the Rho family of GTPases, RhoA, Racl, and cdc42 in SPRY2-mediated inhibition of cell migration and proliferation. The ability of TAT-tagged SPRY2 to inhibit proliferation and migration of IEC-6 cells transfected with constitutively active mutants of RhoA(G14V), Racl(G12V), and cdc42 (F28L) was determined. Constitutively active RhoA(G14V), Racl(G12V), or cdc42(F28L) did not protect cells from the anti-proliferative actions of TAT-SPRY2. The ability of TAT-hSPRY2 to inhibit migration was not altered by of RhoA(G14V) and cdc42(F28L). However, Racl(G12V) obliterated the ability of SPRY2 to inhibit cell autonomous or serum-induced migration. Also, the activation of endogenous Racl was attenuated by TAT-SPRY2. Thus, SPRY2 mediates its anti-migratory actions by inhibiting Racl activation. (C) 2004 Elsevier Inc. All rights reserved.