Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains. Unlike other clostridial binary toxins, binary toxin CDT required exogenous trypsin for activation. Supernatants from all A-B-CDT+ strains caused marked fluid accumulation in the rabbit ileal loop assay after concentration and trypsinization. In addition, the ileal loop response was neutralized by antisera raised against other binary toxin-producing clostridia. Challenge of clindamycin-treated hamsters with these strains resulted in colonization but not diarrhea or death. Binary toxin CDT may play an adjunctive role to toxins A and B in the pathogenesis of C. difficile-associated disease but by itself may not be sufficient to cause disease.