Enterotoxigenic Bacteroides fragilis (ETBF) cells produce a 20-kDa heat-labile metalloprotease toxin which is potentially important in the pathogenesis of diarrhea associated with this infection. Previous studies indicate that subconfluent HT29/C1 cells treated with the B. fragilis toxin (BFT) develop morphologic changes with dissolution of tight clusters and apparent swelling. Such alterations suggest toxin-stimulated reorganization of the cellular cytoskeleton. The purpose of the current study was to evaluate the effect of BFT on actin microfilaments (F-actin) and cell volume. As assessed by fluorescent phallicidin staining which detects F-actin, BFT treatment of HT29/C1 cells resulted in redistribution of F-actin with loss of stress fibers, a floccular staining pattern, and cellular membrane blebbing without quantitative changes in F-actin fluorescence intensity. The F-actin redistribution was time and concentration dependent. In contrast to the cell shrinkage observed in response to the F-actin-depolymerizing agents cytochalasin D and Clostridium difficile toxin A, BFT stimulated an increase in HT29/C1 cell volume of 10 to 25% (compared with control cells) over a 24-h time course. Only 10 to 30 ng of BFT per ml was necessary to stimulate a maximal increase in HT29/C1 cell volume. The effect of BFT on cell volume was persistent and dependent on the proteolytic activity of BFT. In agreement with cell viability assays indicating that BFT did not injure HT29/C1 cells, intoxicated cells exhibited regulatory volume decrease, suggesting that toxin-treated cells remain physiologically dynamic. We conclude that BFT acts on the intestinal epithelial cell cytoskeleton to alter F-actin structure and to stimulate an increase in HT29/C1 cell volume. Although these two activities of BFT appear to be linked, the precise sequence of cellular events following intoxication of HT29/C1 cells with BFT remains unclear. We hypothesize that these F-actin and cell volume changes may lead to an alteration in tight junction function in the polarized intestinal epithelium, contributing to the pathogenesis of diarrhea in ETBF infections.