Matrix metalloproteinase-2 (MMP-2) plays critical roles in cell migration through the breakdown of the extracellular matrix. Cell movements require dynamic actin reorganization, which is controlled by Rho family GTPases. In order to examine the relation between MMP-2 regulation and actin reorganization, we used several inhibitors of Rho family GTPases. Treatment of smooth muscle cells with Clostridium difficile toxin B known to inactivate Rho family GTPases activated MMP-2. However, neither C3 transferase, a Rho inhibitor, nor Y-27632, a specific inhibitor of Rho-kinase, induced MMP-2 activation. Treatment with C3 transferase and Y-27632 caused morphological changes into the round and stellate shape, respectively, by inhibition of actin stress fiber formation. In addition, toxin B treatment induced expression and processing of MT1-MMP, a major activator of MMP-2. Taken together, we suggest the involvement of Rho family GTPases, although inhibition of neither Rho nor Rho-kinase is sufficient, in the activation of MMP-2 through expression and activation of MT1-MMP.