The Rho family GTPases Rac1, RhoA and Cdc42 function as molecular switches that transduce intracellular signals regulating multiple cell functions including gene expression, adhesion, migration and invasion. p53 and its regulator p19Arf, on the other hand, are tumor suppressors that are critical in regulating cell cycle progression and apoptosis. Previously, we have demonstrated that the Rho proteins contribute to the cell proliferation, gene transcription and migration phenotypes unleashed by p19Arf or p53 deletion in primary mouse embryo fibroblasts (MEFs). To further investigate their functional interaction in the present study, we have examined the involvement of Rho signaling pathways in p53-mediated cell invasion. We found that in primary MEFs (1) p53 or p19Arf deficiency led to a marked increase in the number of focal adhesion plaques and fibronectin production, and RhoA, Rac1 and Cdc42 contribute to the p53- and p19Arf-mediated focal adhesion regulation, but not fibronectin synthesis; (2) although endogenous Rac1 activity was required for the p19Arf or p53 deficiency-induced migration phenotype, hyperactive Rho GTPases could not further enhance cell migration, rather they suppressed cell-cell adhesion of p53-/- MEFs; (3) expression of the active mutant of RhoA, Rac1 or Cdc42, but not Ras, promoted an invasion phenotype of p53-/-, not p19Arf-/-, cells; (4) although ROCK activation can partially recapitulate Rho-induced invasion phenotype, multiple pathways regulated by RhoA, in addition to ROCK, are required to fully cooperate with p53 deficiency to promote cell invasion; and (5) extracellular proteases produced by the active RhoA-transduced cells are also required for the invasion phenotype of p53-/- cells. Combined with our previous observations, these results strongly suggest that mitogenic activation of Rho family GTPases can cooperate with p53 deficiency to promote primary cell invasion as well as transformation and that multiple signaling components regulated by the Rho proteins are involved in these processes.