Our previous studies in volunteers immunized with Salmonella enterica serovar Typhi (S. Typhi) have suggested an important role for CD8+ T cells in host defense. In this study we describe a novel subset of nonclassical human HLA-E-restricted S. Typhi-specific CD8+ T cells derived from PBMC of Ty21a typhoid vaccinees. CD3+CD8+CD4-CD56- T cells effectively killed S. Typhi-infected targets regardless of whether they share classical HLA class I molecules with them, by a FAS-independent, granule-dependent mechanism, as evidenced by induction of granzyme B release and the blocking effects of concanamycin and strontium ions. The expression of HLA-E Ags, but not CD1-a, -b, or -c, on the membrane of S. Typhi-infected targets rendered them susceptible to lysis. Moreover, anti-HLA-E Abs partially blocked these responses. We also demonstrated that presentation of S. Typhi Ags via HLA-E could stimulate IFN-gamma production. Increases in the net frequency of IFN-gamma spot-forming cells were observed in the presence of targets coated with peptides that contain S. Typhi GroEL HLA-E binding motifs. These results demonstrate that HLA-E binds nonamer peptides derived from bacterial proteins and trigger CD8+-mediated lysis and IFN-gamma production when exposed to infected targets, raising the possibility that this novel effector mechanism might contribute to host defense against intracellular bacterial infections.