Natural killer (NK) cells are large granular lymphocytes that mediate antigen nonspecific, non-major histocompatibility complex (MHC) restricted lysis of virus infected cells. They are thought to play a role in innate resistance to herpes simplex virus (HSV) infections. In most animal studies reported to date, the virus was injected intraperitoneally, not a natural route of infection. Using a murine model of acute HSV-1 ocular infection, we demonstrate that increased splenic NK activity is induced in BALB/c mice following corneal infection with four different strains of HSV-1. The kinetics of NK cell activation depended on the strain of virus and was associated with virulence of the strain and with the ability of the viruses to grow in vivo. We also assessed the role of interferon-alpha/beta, IFN-gamma, and interleukin-2 (IL-2) in the HSV-1 induced NK cell activation by treating mice with antisera against these lymphokines prior to infection. Treatment with anti-IFN-alpha/beta or anti-IFN-gamma significantly reduced NK cell cytotoxicity, suggesting that these lymphokines were involved in the activation of NK cells following HSV-1 ocular infection. Treatment with anti-IL-2 resulted in increased NK cell activity, suggesting that in vivo, IL-2 is involved in the suppression of NK cell activity in infected mice. Treatment with a combination of anti-IL-2 and anti-IFN also increased NK cytotoxicity. Despite the induction of high levels of NK activity, mice developed severe ocular disease or died of encephalitis.