DX9 mAb-binding killer cell-inhibitory receptors (KIR) recognize HLA-B molecules that express the Bw4 public serologic epitope. We assessed DX9+ NK cell fine specificity recognition of HLA-B7 variants and HLA-B27 alleles by 51Cr release natural cytotoxicity assays and by flow cytometry and enzyme-linked immunospot (ELISPOT) IFN-gamma synthesis and release assays. 721.221 target cell expression of Bw4+ HLA-B27 alleles specifically inhibited DX9+ NK cell natural cytotoxicity and IFN-gamma synthesis and release. A triple substitution of HLA-B7 at residues 80, 82, and 83 known to induce expression of the Bw4 serologic epitope also specifically inhibited DX9+ NK cell natural cytotoxicity and IFN-gamma responses. Single HLA-B7 amino acid substitution variants were recognized in the same decreasing rank order by DX9+ NK cells and Bw4-reactive mAbs: G83R > R82L > N80T = HLA-B7. Natural cytotoxicity inhibition was reversed by the presence of blocking DX9 mAb. Natural cytotoxicity and IFN-gamma production were coordinately regulated by a panel of HLA-B7 variants expressed on 721.221 cells, suggesting that these two effector functions are inhibited by the same KIR-mediated signaling mechanisms. In contrast, some NK cell clones killed 721.221 and K562 target cells equally well but released much more IFN-gamma in response to K562 target cells. Differential regulation of natural cytotoxicity and IFN-gamma release shows that NK cell effector functions respond to distinct signals.