The specificity of human natural killer (NK) cells for herpes simplex virus type 1 (HSV-1)-infected WISH cells was examined by using virus-specific antibodies to block NK killing and by using targets infected with virus variants that express antigenically altered cell surface glycoproteins. Fab fragments of human immunoglobulin (Ig) derived from an HSV-1 seropositive individual blocked NK activity against HSV-1-infected cells, whereas Fab fragments from a seronegative individual had no effect. Fab fragments of monoclonal antibodies specific for different epitopes of the HSV-1 glycoproteins gB and gC blocked NK activity, suggesting that NK cells interact directly with viral glycoproteins. In contrast, a control (HSV-unreactive) monoclonal antibody did not block NK activity. Further evidence that the blocking activity was associated with HSV glycoproteins was obtained by showing that: a) gC-specific antibodies did not decrease NK activity against cells infected with an HSV-1 mutant which does not insert gC into infected cell surface membranes; b) gB-specific antibodies did not reduce NK cytotoxicity against target cells infected with HSV-1 in the presence of 2-deoxy-D-glucose (2dG), which prevents cell surface expression of gB; c) HSV-1 glycoprotein-specific antibodies did not alter NK activity against uninfected cells or against K562, an NK-susceptible tumor cell line; and d) a monoclonal antibody reactive with a cell membrane glycolipid markedly reduced NK reactivity against K562 but had little effect on NK reactivity against HSV-1-infected cells, suggesting that NK cells recognize different determinants on the two types of target cells. Analysis of the fine specificity of NK cell recognition of HSV-1 glycoproteins was performed by using target cells infected with antigenic variants of HSV-1 referred to as monoclonal antibody-resistant (mar) mutants. mar mutants are altered in distinct epitopes of gB or gC, resulting in loss of recognition by the selecting virus-neutralizing monoclonal antibody. NK activity was reduced against cells infected with some, but not all, of these variants. Together, these findings demonstrated a positive correlation between expression of HSV-1 glycoproteins gB and gC and susceptibility of HSV-1-infected target cells to NK activity, implicating the viral glycoproteins as cell surface components recognized by NK cells. Moreover, mutations affecting glycoprotein epitopes defined by monoclonal antibodies also influenced the interaction between NK effector cells and HSV-1-infected target cells, suggesting that viral antigenic sites recognized by NK cells overlap with those recognized by anti-HSV-1 antibodies.