Rho small G protein regulates various actin-dependent cell functions. As to the functioning sites of Rho, Rho regulates formation of stress fibers and focal adhesions in many types of cultured cells, whereas we have shown that the association sites of actin filaments with the plasma membrane controlled by the ERM (Ezrin, Radixin, Moesin) family are the functioning sites of Rho in MDCK cells stably expressing myc-RhoA. We have investigated here the effect of microinjection of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, or guanosine 5'-(3-O-thio) triphosphate-bound active form of Rho on the intracellular localization of both the ERM family and vinculin, which is one of the structural proteins of focal adhesions, in wild type MDCK cells. The ERM family was preferentially localized at peripheral bundles of actin filaments which are localized at the outer edge of colonies of the cells, microvilli and low Ca2+-induced cortical bundles of actin filaments in wild type MDCK cells. Microinjection of Rho GDI or C3 inhibited the localization of the ERM family at both the peripheral bundles and the low Ca2+-induced cortical bundles. On the other hand, vinculin was localized at both focal adhesions and basal edges of the colonies of the cells, and microinjection of Rho GDI or C3 inhibited the localization of vinculin at both of these sites. These results indicate that activation of Rho is necessary for the association of both the ERM family and vinculin with the plasma membrane in wild type MDCK cells. Microinjection of the guanosine 5'-(3-O-thio) triphosphate-bound form of Rho induced an increase in the localization of vinculin at focal adhesions, but did not induce an increase in the localization of the ERM family at the plasma membrane, indicating that activation of Rho itself is sufficient only for the association of vinculin with the plasma membrane at focal adhesions.