It has previously been shown that the plasmid-encoded YopE protein of Yersinia pseudotuberculosis is a virulence determinant. In this study, HeLa cells, macrophages and mice were used as different model systems to determine the actual role of YopE in the virulence process. The YopE protein mediates a cytotoxic response on a confluent layer of HeLa cells. A prerequisite of this activity is that the pathogen binds to the cell surface. YopE also induces a cytotoxic response on mouse macrophages where it influences the ability of the pathogen to resist phagocytosis. Bacterial mutants defective in their ability to express YopE are avirulent after oral or intraperitoneal infection but virulent following intravenous injection. On the basis of these results, we propose a role for YopE in the virulence process of Yersinia.