The endothelial cytoskeleton is important for the regulation of endothelial barrier function. Small GTP-binding Rho proteins play a central role in the organization of the microfilament system. Clostridium difficile toxin B (TcdB) inactivates Rho proteins by glucosylation at Thr-37. We used TcdB as a probe to study the role of Rho proteins in the regulation of endothelial barrier function. TcdB time (50-170 min) and dose (10-100 ng/ml) dependently increased the hydraulic conductivity of cultured porcine pulmonary artery endothelial cell monolayers approximately 10-fold. Simultaneously, the albumin reflection coefficient decreased substantially from 0.8 to 0.15. Before endothelial hyperpermeability, TcdB reduced F-actin content in a dose-dependent manner, whereas G-actin content remained unchanged. Finally, we proved that TcdB caused dose (5-100 ng/ml)- and time-dependent glucosylation of Rho proteins in endothelial cells. Phalloidin, which stabilizes filamentous actin, prevented the effect of TcdB on endothelial permeability. In contrast to thrombin-, hydrogen peroxide-, or Escherichia coli hemolysin-induced hyperpermeability, the elevation of cyclic nucleotides did not block TcdB-related permeability. The data demonstrate a central role of small GTP-binding Rho proteins for the control of endothelial barrier function.