Rho family GTPases are known to be involved in cytoskeletal reorganization. We examined the possibility that these functions may be dictated by a balance of Rho family GTPase signaling. Using transient viral expression of RhoA, Rac1, Cdc42 and their mutants, as well as C3 exoenzyme, we altered cytoskeletal organization under normal growth conditions. Overexpression of wild-type or constitutively active forms of the Rho family GTPases led to their respective activation phenotypes. Overexpression of dominant negative forms of given Rho family GTPases led to a phenotype consistent with activation of the other Rho family GTPase. Treatment with C. difficile toxin A, that inactivates all Rho family GTPases, led to the transient appearance of a variety of activation phenotypes. Previously, we reported that inactivation of Rho led to induction of apoptosis, implying that Rho may play an important role in cell survival signaling. This signaling, however, is not affected by expression of any forms of Rac1 or Cdc42, and only inactivation of Rho led to induction of apoptosis. Rho family GTPases appear to coordinate cytoskeletal organization by a balance of signaling, while cell survival is regulated by a distinct Rho-mediated signaling pathway.