The effects of the C. difficile toxin were examined in [human cervical carcinoma] HeLa cells and in mouse adrenal tumor (MAT) cells. Cytotoxicity was evaluated by vital dye exclusion and 51Cr release. In HeLa and MAT cells, C. difficile toxin caused rounding of virtually 100% of cells. This rounding was distinguishable from rounding produced by the Escherichia coli heat labile enterotoxin (LT). LT was inactive in HeLa cells; in MAT cells, C. difficile toxin produced uniformly rounded cells, while LT rounded cells usually had cytoplasmic extensions and a regular background of flattened cells. In C. difficile toxin affected HeLa cells, there were < 2 mitotic figures per 1000 cells compared with 15-18 in controls. C. difficile toxin 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 in C. difficile toxin 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 wk cloning assay in which C. difficile toxin treated cells had a cloning efficiency of < 1% compared with .apprx. 10% in controls. Thus, the major effect of the C. difficile toxin evidently is inhibition of growth rather than immediate cell death. The relationship of this growth inhibition to colitis remains to be elucidated.