Small GTP-binding proteins of the Ras and Rho family participate in various important signalling pathways. Large clostridial cytotoxins inactivate GTPases by UDP-glucosylation. UsingClostridium difficiletoxin B-10463 (TcdB) for inactivation of Rho proteins (RhoA/Rac/Cdc42) andClostridium sordelliilethal toxin-1522 (TcsL) for inactivation of Ras-proteins (Ras/Rac/Ral, Rap) the role of these GTPases in protein kinase C (PKC) stimulation was studied. Phorbol-myristate-acetate (PMA) induced a rapid PKC translocation to and activation in the particulate cell fraction as determined by PKC-activity measurements and Western blots for PKCα. These effects were blocked by TcdB inhibiting Rho proteins in endothelial cells, but not in TcsL-treated cells (i.e., cells without Ras activity), suggesting that Rho GTPases (RhoA and/or Cdc42) are the most likely GTP-binding proteins responsible for PKC activation. The Rho requirement for PKC activation/translocation was also verified for human epithelial cells and for lipopolysaccharide-stimulated endothelial cells. In summary, the data presented indicate that Rho protein inhibition blocked PKC translocation/activation in endothelial and epithelial cells.