The presence of natural killer (NK) cells contributes to early defense against murine cytomegalovirus (MCMV) infection. Although NK cells can mediate in vivo protection against MCMV, the mechanism by which they do so has not been defined. The studies presented here evaluate cytokine production by NK cells activated during MCMV infection and the role of NK cell-produced cytokines in early in vivo antiviral defenses. Experiments with normal C57BL/6, T cell-deficient C57BL/6 nude, and severe combined immunodeficient mice lacking T and B cells demonstrated that both interferon gamma (IFN-gamma) and tumor necrosis factor (TNF) production were induced at early times after infection with MCMV. Conditioned media samples prepared with cells from these mice, on day 2 after infection, produced 11-43 pg/million cells of IFN-gamma and 12-19 pg/million cells of TNF as evaluated by specific protein enzyme-linked immunosorbent assays. Studies in the NK- and T cell-deficient mouse line, E26, in mice that had been depleted in vivo of NK cells by treatment with antibodies eliminating NK cells, anti-asialo ganglio-N-tetraosylceramide or anti-NK1.1, and with populations of cells that had been depleted of NK cells by complement treatment with the anti-NK cell antibody, SW3A4, demonstrated that NK cells were solely responsible for the IFN-gamma but were not required for TNF production. The in vivo absence of NK cells was accompanied by increased viral hepatitis and viral replication in both immunocompetent and immunodeficient mice, as well as decreased survival time of immunodeficient mice. In vivo treatments with antibodies neutralizing IFN-gamma demonstrated that this factor contributed to the NK cell-mediated antiviral defense and reduced the measured parameters of viral defense to levels indistinguishable from those observed in NK cell-deficient mice. These effects appeared to be independent of cytolytic activity, as NK cells isolated from anti-IFN-gamma-treated mice mediated killing at levels comparable to those observed in control-treated mice. The consequences of interleukin 12 (IL-12) administration, a known potent inducer of IFN-gamma production by NK cells, were evaluated in MCMV-infected mice. Low IL-12 doses, i.e., 1 ng/d, increased NK cell cytotoxicity and IFN-gamma production up to twofold and resulted in improved antiviral status; virus-induced hepatitis was decreased as much as fivefold, and viral burdens were decreased to levels below detection.(ABSTRACT TRUNCATED AT 400 WORDS)