Clostridium difficile toxin B is a powerful cytopathic agent which causes animal cells in culture to become rounded and arborized, an effect similar to that induced by the cytochalasins. In this study, we demonstrated that the morphological effects of the toxin are directed specifically against the actin and related components of the cytoskeleton. Dramatic disruption and reorganization of the actin stress fibers were detectable prior to significant changes in cell shape and alterations in the microtubular and intermediate filament networks. Along with F-actin, the adhesion plaque proteins, vinculin and talin were localized in intoxicated cells in a patchy pattern reminiscent of that seen in cells treated with phorbol esters or transformed by oncogenic viruses. A quantitative fluorescence assay for cellular F-actin showed that these morphological changes were accompanied by a modest net depolymerization of only 15 to 20% of the actin filaments in the cell, and that depolymerization was closely correlated with changes in cell shape. In complementary studies on cells spreading on a substrate, we found that the toxin affected the actin content and the shape of the processes extended from the cell body. As in cells treated with cytochalasin, there was a differential response between normal and virally transformed cells spreading in the presence of the toxin. The results of this study support the view that C. difficile toxin B affects one or more cellular components that regulate the structure and function of the actin cytoskeleton, and that its predominant effect is to cause a dramatic disruption of stress fibers and relocalization of the F-actin.+