The pathogenic bacterium Actinobacillus actinomycetemcomitans expresses a leukotoxin (Ltx) and cytolethal distending toxin (CDT) with cytolytic properties. CDT also has cytostatic properties, inducing a G cell cycle block. The extent of the contribution of these, as well as other toxins, to the cytolytic and cytostatic activities of this microorganism have not been defined and the aim of this study was to determine their contribution. To that end, a naturally transformable A. actinomycetemcomitans clinical strain (D7S-smooth) was used to construct a series of deletion mutants (ΔcdtA, ΔcdtB, ΔcdtC, ΔcdtABC, ΔltxA, ΔltxA/Δ cdtABC). Human peripheral blood mononuclear cells were incubated with cell-associated and extracellular bacterial preparations. The ability of wild type and isogenic mutants to induce T-cell apoptosis and cell cycle arrest was compared. The expression of ltxA and each of the cdt gene loci partially contributed to A. actinomycetemcomitans apoptosis, since each of the isogenic mutants exhibited reduced ability to induce T-cell apoptosis. Conversely, the ability to induce cell cycle block was abolished in each of the cdt isogenic mutants. A mutant with simultaneous deletion of ltxA and cdtABC genes retained potent ability to induce apoptosis in its cell-associated, but not extracellular, preparation. Neutralization with Escherichia coli anti-GroEL monoclonal antibody, lead to significant diminution of apoptosis-inducing activity of the ΔltrA/ΔcdtABC cell-associated preparation. These data provide evidence for the expression of other A. actinomycetemcomitans cytolytic molecule(s) distinct from CDT and leukotoxin, with a possible role for GroEL-like molecule in T-cell apoptosis.