Aggregatibacter actinomycetemcomitans strains that express cytolethal distending toxin (Cdt) are associated with localized aggressive periodontitis. However, the in vivo targets of Cdt in the human oral cavity have not been firmly established. Here, we demonstrate that A. actinomycetemcomitans Cdt kills proliferating and nonproliferating U937 monocytic cells at a comparable specific activity, approximately 1.5-fold lower than that against the Cdt-hypersensitive Jurkat T-cell line. Cdt functioned both as a DNase and a phosphatidylinositol 3-phosphate (PIP(3)) phosphatase, and these activities were distinguished by site-specific mutagenesis of the active site residues of CdtB. Using these mutants, we determined that the DNase activity of CdtB is required for cell cycle arrest and caspase-dependent induction of apoptosis in proliferating U937 cells. In contrast, Cdt holotoxin induced apoptosis by a mechanism independent of caspase- and apoptosis-inducing factor in nonproliferating U937 cells. Furthermore, apoptosis of nonproliferating U937 cells was unaffected by the Cdt mutant possessing reduced phosphatase activity or by the addition of a specific PIP(3) phosphatase inhibitor, suggesting that the induction of apoptosis is independent of phosphatase activity. These results indicate that Cdt intoxication of proliferating and nonproliferating U937 cells occurs by distinct mechanisms and suggest that macrophages may also be potential in vivo targets of Cdt.