Lymphocytic choriomeningitis virus (LCMV) infection of C3H/St, nude (BALB/c background), and other mice induced high levels of natural killer (NK) cell activity in the spleen and peritoneum. L-929 cells were used as targetsand were not lysed by spleen or peritoneal cells from uninfected mice. The cytotoxic cells were characterized as NK cells because they were nonadherent, nonphagocytic lymphocytes lacking theta and immunoglobulin antigens on their plasma membranes. Their activity was sensitive to 6 mM EDTA and to heating for 5 h at 37 degrees C, but resisted treatment with 0.5 percent trypsin. No role for antibody could be demonstrated in these assays. Relative to cytotoxic T-cell activity, the induction of NK cell activity was resistant to X-irradiation of mice with 1,000 rads but was sensitive if mice were first treated with Strontium-89, a bone-seeking isotope. NK cells were induced by LCMV in all tested strains of mice. In C3H/St mice NK cell activity was detected as early as 1 day and peaked at 3 days postinfection. Maximum activity in C3H/St mice was observed in mice 5-10 wk of age, but significant NK activity was also induced in newborns, which subsequently carried virus in their tissues for the duration of their lives. Older LCMV-carriers did not have detectable spleen NK cell activity. No memory oranamnestic response could be demonstrated for NK cell induction. NK cell activity was not induced by LCMV challenge of LCMV-immune mice, but was induced in those mice by infection with Pichinde virus, a closely related virus. The advent of NK cell activity correlated with the synthesis of interferon in LCMV-infected mice. Culture fluids lacking virus infectivity but containing interferon induced cytotoxic cell activity in nude and C3H/St mice. These experiments suggest that LCMV induced NK cells via an interferon-dependent mechanism. When studied in several strains of mice, the continued expression of NK cell activity did not seem to directly correlate with spleen interferon levels, suggesting that additional factors may play a role as well in maintaining the activity of the NK cell in vivo.