Pseudomonas aeruginosa is an opportunistic bacterial pathogen that primarily infects immunocompromised individuals and patients with cystic fibrosis. Using a tissue culture system, invasive strains of P. aeruginosa were discovered to induce apoptosis at high frequency in HeLa and other epithelial and fibroblast cell lines. This apoptotic phenotype in the infected cells was determined by several criteria including (i) visual changes in cell morphology, (ii) induction of chromatin condensation and nuclear marginalization, (iii) the presence of a high percentage of cells with subG1 DNA content, and (iv) activation of caspase-3 activity. Induction of the type III secretion machinery, but not invasion of P. aeruginosa is required for induction of apoptosis. The apoptosis phenotype is independent of the cytoskeletal rearrangements that occur in the host cell early after infection. Mutants in P. aeruginosa exoS fail to induce apoptosis and complementation with wild-type exoS restored the apoptosis-inducing capacity, demonstrating that ExoS is the effector molecule. Analysis of exoS activity mutants shows that the ADP-ribosylating capacity of ExoS is essential for inducing the apoptotic pathway.