Yersinia pseudotuberculosis is a pathogenic enteric bacteria that evades host cellular immune response and resides extracellularly in vivo. Nevertheless, an important contribution of T cells to defense against Yersinia has been previously established. In this study we demonstrate that Lewis rats infected with virulent strains of Y. pseudotuberculosis, mount a Yersinia-specific, RT1-A-restricted, CD8+ T cell-mediated, cytotoxic response. Sensitization of lymphoblast target cells for cytolysis by Yersinia-specific CTLs required their incubation with live Yersinia and was independent of endocytosis. Although fully virulent Yersinia did not invade those cells, they attached to their surface. In contrast, invasin-deficient strain failed to bind to blast targets or to sensitize them for cytolysis. Furthermore, an intact virulence plasmid was an absolute requirement for Yersinia to sensitize blast targets for cytolysis. Using a series of Y. pseudotuberculosis mutants selectively deficient in virulence plasmid-encoded proteins, we found no evidence for a specific role played by YadA, YopH, YpkA, or YopJ in the sensitization process of blast targets. In contrast, mutations suppressing YopB, YopD, or YopE expression abolished the capacity of Yersinia to sensitize blast targets. These results are consistent with a model in which extracellular Yersinia bound to lymphoblast targets via invasin translocate inside eukaryotic cytosol YopE, which is presented in a class I-restricted fashion to CD8+ cytotoxic T cells. This system could represent a more general mechanism by which bacteria harboring a host cell contact-dependent or type III secretion apparatus trigger a class I-restricted CD8+ T cell response.