The contribution of toxin B to Clostridium difficile-associated infection is undefined. Toxin B induces dramatic phenotypic alterations (cytotoxic effects) in cultured mesenchymal and nonintestinal epithelial cells, yet its effects on intestinal epithelial cells are not clearly understood. The alterations induced by toxin B in nonintestinal cells appear to be secondary to toxin-induced redistribution of filamentous actin. It has not been determined whether toxin B exerts similar effects on cultured intestinal epithelial cells or whether such phenotypic alterations are of any physiological consequence. To address these questions, we examined the effect of C. difficile toxin B on the phenotype and barrier function of T84 cell monolayers. Our studies show that the cytotoxic effects of toxin B, i.e., cell rounding, do extend to cultured intestinal epithelial cells (T84). In addition, toxin B dramatically reduces the barrier function of T84 monolayers grown on collagen-coated filters. Toxin B-induced redistribution of filamentous actin appears to be responsible for the alterations in both intestinal epithelial cell phenotype and barrier function. Specifically, filamentous actin comprising the perijunctional actomyosin ring, known to be important in regulating tight junction permeability, is condensed into discrete plaques. Flux studies confirm that the permeability defect is at the level of the tight junction. We conclude that toxin-induced changes in actin distribution perturb intercellular junctional contacts and thereby ablate epithelial barrier function. There was no evidence of cell death as determined by lactate dehydrogenase release assays.