Cytolethal distending toxin (CDT) from Actinobacillus actinomycetemcomitans is a G2/M cell-cycle-specific growth-inhibitory toxin that leads to target cell distension followed by cell death. To determine the mechanisms by which A. actinomycetemcomitans CDT acts as an immunosuppressive factor, we examined the effects of highly purified CDT holotoxin on human T lymphocytes. Purified CDT was cytolethal toward normal peripheral T lymphocytes that were activated by in vitro stimulation with phytohemagglutinin. In addition, purified CDT showed cytolethal activity against Jurkat and MOLT-4 cells, which are known to be sensitive and resistant, respectively, to Fas-mediated apoptosis. Death in these cell lines was accompanied by the biochemical features of apoptosis, including membrane conformational changes, intranucleosomal DNA cleavage, and an increase in caspase activity in the cells. Pretreatment of Jurkat cells with the general caspase inhibitor z-VAD-fmk mostly suppressed CDT-induced apoptosis. Furthermore, specific inhibitors of caspase-2 and -7 showed significant inhibitory effects on CDT-induced apoptosis in Jurkat cells, and these inhibitory effects were fully associated with reduced activity of caspase-2 or -7 in the CDT-treated Jurkat cells. These results strongly suggest that CDT possesses the ability to induce human T-cell apoptosis through activation of caspase-2 and -7.