Human NK cells bear surface receptors that inhibit their cytolytic activity upon specific recognition of MHC class Ia Ags; little is known about the capacity of class Ib molecules to regulate NK cell function. We have studied the roles of different NK inhibitory receptors in recognition of the class Ib HLA-G. To this end, we analyzed the ability of an HLA-defective tumor cell line (721.221) transfected with the membrane form of HLA-G1, which contains the three external domains, to inhibit the cytolytic activity mediated by a panel of NK clones from several donors. A substantial proportion of peripheral blood NK clones appeared to be significantly inhibited by the HLA-G1-transfected cell line (referred to as .221-G1); nevertheless, no relation was observed between the expression and the function of serologically identifiable Ig-SF receptors (p58/p70) and specific recognition of .221-G1 cells. Moreover, p58 killer cell inhibitory receptor-IgG soluble fusion proteins specifically bound to 721.221 transfectants bearing their corresponding HLA-C ligands, but only a weak reactivity with .221-G1 cells was detectable. By contrast, most NK clones blocked by HLA-G1 expressed the CD94/NKG2-A inhibitory receptor, and moreover, CD94-specific mAbs reconstituted their cytolytic activity comparably to anti-HLA class I mAbs. These data support the idea that the CD94/NKG2 receptor complex is involved in the recognition of cells expressing HLA-G1.