The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of α-Toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified α-Toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two α-Toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce α-Toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of α-Toxin during endothelial cell invasion by S. aureus enhances apoptosis.