Cytotoxic necrotizing factor 1 (CNF1), a 110-kDa protein toxin from pathogenic Escherichia coli induces actin reorganization into stress fibers and retraction fibers in human epithelial cultured cells allowing them to spread. CNF1 is acting in the cytosol since microinjection of the toxin into HEp-2 cells mimics the effects of the externally applied CNF1. Incubation in vitro of CNF1 with recombinant small GTPases induces a modification of Rho (but not of Rac, Cdc42, Ras, or Rab6) as demonstrated by a discrete increase in the apparent molecular weight of the molecule. Preincubation of cells with CNF1 impairs the cytotoxic effects of Clostridium difficile toxin B, which inactivates Rho but not those of Clostridium sordellii LT toxin, which inhibits Ras and Rac. As shown for Rho-GTP, CNF1 activates, in a time- and dose-dependent manner, a cytoskeleton-associated phosphatidylinositol 4-phosphate 5-kinase. However, neither the phosphatidylinositol 4,5-bisphosphate (PIP2) nor the phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) or 3,4,5-trisphosphate (PIP3) cellular content were found increased in CNF1 treated HEp-2 cells. Cellular effects of CNF1 were not blocked by LY294002, a stable inhibitor of the phosphoinositide 3-kinase. Incubation of HEp-2 cells with CNF1 induces relocalization of myosin 2 in stress fibers but not in retraction fibers. Altogether, our data indicate that CNF1 is a toxin that selectively activates the Rho GTP-binding protein, thus inducing contractility and cell spreading.