Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice. exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB Mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticits Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticits CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB Mutation. These results suggest that the effect of H. hepaticits CDT probably involves the DNase activity of CdtB. (C) 2004 Elsevier Inc. All rights reserved.