Major histocompatibility complex (MHC) class I molecules induce inhibitory signals on natural killer (NK) cells via killer cell immunoglobulin-like receptors (KIR). We recently reported a human single-chain antibody (scFv#1), which recognizes an epitope on HLA-Cw6 (genotype: *0602). Flow cytometry showed scFv#1 binding to HLA-Cw6 (strong) and also to HLA-Cw2, 4, 5 (very weak) but not to HLA-Cw1, 3, 7, 8. The presumptive epitope of the antibody fragment, which includes residues Asn77 and Lys80 was verified by introducing point mutations into HLA-Cw6 encoding cDNAs. Asn77 --> Ser77 (N77S) and Lys80- -> Asn80 (K80N) mutants of Cw6 lost scFv#1 binding capacity whereas an additional mutation at aa position 90 (Asp-->Ala, D90A) did not influence scFv#1 binding characteristics. Since residues 77 and 80 of HLA-C are directly involved in KIR/MHC interaction, we expected the induction of target cell lysis upon addition of scFv#1 when bringing NK and HLA-Cw6 positive cells together. To prove this interference, we performed Cr-release assays, using Cw*0602 and mock-transfected K562 erythroleukemia cells as targets and freshly prepared peripheral blood NK cells as effector cells. scFv#1 appeared to influence KIR on ligand binding and restored lysis at low effector to target (E/T) ratios. Pan HLA class I antibody W6/32 did not show such effects. Taken together scFv#1 binding patterns with mutagenized HLA-Cw6 and Cr-release assays are strong evidence that the scFv#1 epitope on HLA-Cw6 is at or close to the binding site of CD158a.