Natural killer (NK) cells have been implicated in the recognition and killing of a variety of virus infected target cells in vitro, yet their role in vivo remains uncertain. In these experiments, the role of NK cells in the regulation of resistance to herpes simplex virus-1 (HSV-1) was studied. Adult C57BL/6 mice are resistant to HSV-1 (HFEM strain), but are rendered highly susceptible by treatment with cyclophosphamide 24 hr prior to infection. In this model, passive transfer of 10(8) normal spleen cells or 10(7) poly I:C-treated spleen cells provided protection for 72% of the recipients. Spleen cells from NK cell-deficient beige mice similarly treated failed to engender passive protection. The phenotype of the cells responsible for transferring protection was NK1.1+, and asialo GM1+. Transfer of NK cells resulted in marked reduction of HSV titers in the livers and brains of recipients. These experiments provide direct evidence for a role for NK cells in protection against development of fatal HSV infection in mice.