Human natural killer (NK) cells were shown to be much more cytolytic for WISH cells infected with herpes simplex virus type 1 (HSV-1) than for uninfected cells during 18-hour 51Cr release assays. Cold-target competition experiments involving high cold target to radiolabeled target cell ratios demonstrated that infected cells specifically competed for lytic activity against infected cell targets, and, therefore, the infected cells were not inherently more sensitive to lysis than uninfected cells. In contrast to these findings, depletion experiments involving low ratios of cold HSV-1-infected targets to labeled infected target cells resulted in increased lytic activity against uninfected cell targets. This finding suggested that NK effectors with specificity for WISH cell surface determinants had become highly activated during the depletion incubation. The addition of interferon alpha and particularly interleukin 2 to NK cytotoxicity assays enhanced NK cytolytic activity against both infected and uninfected target cells in a dose-dependent manner. Interleukin 1 did not give this effect. However, the enhanced lytic activity of NK cells following exposure to low doses of infected cell cold targets cannot be explained solely on the basis of release of the three lymphokines tested, since interleukin 1 was not effective, interleukin 2 was not detected in culture supernatants derived from the competition experiments, and NK cells preferentially lyse HSV-1-infected target cells independent of the enhancing effects of interferon. Together, the results indicated that NK cells recognize and bind to specific target cell surface structures which may, in turn, enhance their lytic activity.