It is projected that in 2005, approximately 220 900 men will be newly diagnosed with carcinoma of the prostate (CaP). Men who are diagnosed with locally advanced or metastatic disease undergo androgen ablation therapy and most will relapse and progress within 18 months. Metastasis to bone is the major clinical concern during CaP progression, as it is associated with intractable pain, bone fracture and paralysis resulting from spinal cord compression. Therefore, an understanding of the key mechanisms involved in CaP cell bone metastasis is vital to development of novel treatments. The Rho GTPases are molecular switches involved in cell survival, motility and invasion. Increased expression of RhoC GTPase is linked to enhanced metastatic potential in multiple cancers; however, the role of RhoC GTPase in CaP metastasis has not been addressed. In the current study, we demonstrate that RhoC GTPase is expressed and active in PC-3 CaP cells. RhoC inhibition, either pharmacologically with C3 exotransferase or molecularly through expression of a dominant-negative RhoC, promotes IGF-I stimulated random motility but decreases in vitro invasion and experimental metastases. Inhibition of RhoC activity results in drastic morphologic changes and alterations in the expression and distribution of focal adhesion-related proteins. These data suggest that RhoC inhibition leads to activation of other GTPases involved in nondirected motility and that expression of active RhoC is required for the invasive phenotype of PC-3 cells.