Previous studies on NK recognition of HLA‐G1 employed as targets 721.221 transfectants (.221‐G1) that unknowingly co‐expressed the HLA‐E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re‐evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA‐G1 by NK clones. We employed as targets .221‐G1 cells and a surface HLA‐E‐negative transfectant, .221‐G1(E_neg), generated by site‐directed mutagenesis of the HLA‐G1 leader sequence. The antagonistic effects of receptor‐ (i.e. CD94/NKG2A, ILT2) and ligand‐specific mAb (i.e. HLA‐G, HLA‐E) were assessed. In addition, binding of an ILT2‐Ig fusion protein to the .221‐AEH, expressing only HLA‐E, and the .221‐G1(E_neg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA‐G1 involves at least two non‐overlapping receptor‐ligand systems: the CD94/NKG2 interaction with HLA‐E, and the engagement of the ILT2(LIR1) receptor by HLA‐G1 molecules.