The engagement of the activating isoforms of C-type lectin inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I non-A, -B, -C, and -G) or sHLA-I–Cw4 or –Cw3 from untransfected or –Cw4 or –Cw3 alleles transfected HLA-A⁻, B⁻, C⁻, G⁻, E⁺, F⁺ 721.221 lymphoblastoid cell line, respectively, was blocked by covering the corresponding activating receptor with either anti-CLIR– or anti-KIR–specific monoclonal antibodies (mAbs). After sHLA-I–activating receptor interaction, NK cells produced and released Fas ligand (FasL), which in turn led to NK cell apoptosis by interacting with Fas at the NK cell surface. Blocking anti-Fas mAb, or anti-FasL mAb, inhibited sHLA-I–mediated apoptosis via activating receptor in NK cell clones. This apoptosis was inhibited by NK cell treatment with cyclosporin A, whereas this drug had no effect on activating receptor–mediated activation of cytolysis. Conversely, concanamycin A, an inhibitor of vacuolar type H⁺–adenosine triphosphatase (H⁺-ATPase) of granules, inhibited activating receptor–induced NK cell cytolysis, suggesting that activating receptor–mediated apoptosis and cytolysis can use different intracellular pathways. Furthermore, a large amount of interferon-γ (IFN-γ) was detectable in culture supernatant of activating receptor⁺ NK cells incubated with the appropriate sHLA-I ligand. Again, cyclosporin A, but not concanamycin A, strongly reduced activating receptor–mediated IFN-γ production. This suggests that activating receptor–induced apoptosis of NK cells could play a role in eliminating potentially harmful NK cell clones and, at the same time, it leads to production of IFN-γ, an antiviral cytokine able to amplify immune responses.