HLA-E-restricted T cells represent a minor cytolytic T lymphocyte (CTL) population characterized by the surface expression of HLA class I-specific inhibitory receptors and by the capability of killing a large panel of allogeneic target cells (therefore named NK-CTL). Here we show that this subset of T cells is present in a sizeable fraction in the peripheral blood of human cytomegalovirus (HCMV)-seropositive healthy individuals. We provide evidence that NK-CTL recognize in an HLA-E-restricted fashion a naturally processed CMV-derived peptide in the transporter associated with antigen processing (TAP)-2–/– UL40+ RMA-S cell transfectants. Moreover, we show that they recognize and kill HCMV-infected target cells. NK-CTL are characterized by the CD8βdull CD45RA+CD28–CD27–CCR7–CD56+ surface phenotype, thus suggesting that they belong to the effector-memory cell compartment. Consistent with the effector-memory phenotype, they promptly produce IFN-γ, but not IL-2, upon interaction with the specific HCMV UL40-derived peptide. Our data suggest that HLA-E-restricted CTL may represent an additional effector cell type involved in defenses against HCMV, a virus which escapes the control exerted by conventional CTL or NK cells.