The small GTP-binding protein Rho regulates the assembly of actin stress fibers and focal adhesions in cells responding to growth factors. ADP-ribosylation of Rho by C3 transferase blocks this function; however, an enzymatic target for Rho has not yet been defined. We now report that Rho activates phosphatidylinositide 3-kinase in soluble preparations of platelets. Activation of phosphatidylinositide 3-kinase by GTP gamma S is blocked by ADP-ribosylation of endogenous Rho, and Rho shifts to the cytoskeleton in platelets exposed to thrombin. The inhibitory effects of ADP-ribosylation are overcome by exogenous recombinant Rho but not by recombinant Rac, another member of the Ras superfamily. Exposure of platelets to thrombin has been reported to lead to activation of phosphatidylinositide 3-kinase, a shift of this enzyme to the platelet membrane skeleton, and rapid cytoskeletal reorganization. In other studies, ADP-ribosylation of Rho has been found to inhibit thrombin-induced platelet aggregation, a cytoskeletally linked event. We suggest that Rho may exert its effects on cytoskeletal reorganization via phosphatidylinositide 3-kinase.