Cells normally insensitive to human natural killer (NK) activity were rendered susceptible by infection with HSV-1. The cytotoxic effector cell was a nonadherent, non-T, non-B lymphocyte. Antibody plus complement treatment, using a monoclonal antibody that recognizes an antigen present on NK cells, removed much of the cytotoxic activity, and a density gradient fraction enriched for NK cells yielded cells of increased virus-specific cytotoxicity. It was concluded that the effector cell active against infected targets possessed characteristics of an NK cell. Blockage of viral protein synthesis during infection inhibited development of increased susceptibility of infected targets to NK activity. When targets were infected with a mutant virus unable to produce viral glycoprotein C (gC), NK activity against these targets was reduced approximately 30% compared with activity against targets infected with wild-type virus. Similarly, activity against targets infected in the presence of 2-deoxyglucose (2dG), which prevents cell surface expression of viral glycoprotein B (gB), was also reduced approximately 30%. An approximately 60% reduction in activity was seen against targets infected with mutant virus in the presence of 2dG; these targets express gD, but neither gB nor gC. When cells expressing various combinations of HSV-1 glycoproteins were used as both labeled targets and cold target competitors, it was found that the susceptibility of a particular target to NK activity was paralleled by its ability to act as a cold target competitor. This indicates that targets with decreased sensitivity to NK cells were less able to bind NK effectors. Further, the amount of interferon produced in co-cultures of NK effectors and infected target cells did not directly correlate with the amount of NK activity generated, and interferon pretreatment of effectors did not decrease virus-specific cytotoxicity. The present results suggest that HSV-1 glycoproteins expressed at the surface of infected targets may act as recognition structures for NK cells.