Toxins A and B of Clostridium difficile are large cytotoxic proteins that share several unusual structural features, including four conserved cysteines, a potential nucleotide binding site, a hydrophobic region, and a series of contiguous repeating units at the carboxyl terminus. In the following study, we developed a series of toxin B mutants with altered properties in each of these features and examined the effect of the mutation on cytotoxic activity. Altering conserved cysteines to serine resulted in a 90% reduction in activity, whereas altering a histidine residue located in the potential nucleotide binding site to glutamine resulted in a 99% reduction. Removing the repeating units lowered the activity by 90% whereas removing the repeating units plus a conserved cysteine located just upstream of the units reduced the activity by more than five logs, resulting in an inactive toxin. Deleting the internal hydrophobic region had a similar effect. Our findings demonstrate that these conserved features appear to be important for expression of cytotoxic activity.