The effects of theClostridium difficiletoxin were examined in HeLa and mouse adrenal tumor (MAT) cells. Cytotoxicity was evaluated by vital dye exclusion and51Cr release. In both HeLa and MAT cells,C.difficiletoxin caused rounding of virtually 100% of cells. This rounding was distinguishable from rounding produced by theEscherichia coliheat-labile enterotoxin (LT): (1) LT was inactive in HeLa cells; (2) in MAT cells,C.difficiletoxin produced uniformly rounded cells, while LT-rounded cells usually had cytoplasmic extensions and a regular background of flattened cells. InC.difficiletoxin affected HeLa cells, there were <2 mitotic figures per 1000 cells compared with 15–18 in controls.Clostridium difficiletoxin treated HeLa cells showed <10% cytotoxicity in 24 h and no more than 30% by 74 h, similar to control cells. However, paralleling the suppression of mitotic figures, cell multiplication was inhibited inC.difficiletoxin treated cells when subcultured in a short-term (72 h) assay compared with up to a 500% increase in control cells. This inhibition was also seen in a 6-week cloning assay in whichC.difficiletoxin treated cells had a cloning efficiency of <1% compared with approximately 10% in controls. We conclude that the major effect of theC.difficiletoxin is inhibition of growth rather than immediate cell death. The relationship of this growth inhibition to colitis remains to be elucidated.