The immunological mechanisms involved in virus-induced hepatitis were examined by measuring the cytotoxic capabilities and the morphological and antigenic phenotypes of leukocytes isolated from the livers of virus-infected mice. Large granular lymphocytes (LGL) of both natural killer (NK) cell and cytotoxic T lymphocyte (CTL) phenoytpes [phenotypes] accumulated in livers of mice infected with lymphocytic choriomeningitis virus (LCMV) of either the nonhepatotropic Armstrong strain (LCMV-ARM) or the hepatotropic WE strain (LCMV-WE). NK cell activity and LGL number increased 3- to 4-fold between days 1 and 5 postinfection (p.i.). These LGL were characterized as NK cells on the basis of cell surface antigens, kinetics of appearance, target cell range, and morphology. By day 7 p.i., virus-specific, H-2-restricted, Thy-1+, Lyt-2+CTL activity was present in the liver, and its appearance correlated with a second wave of LGL accumulation. Total CTL activity, leukocyte numbers, and CTL/LGL numbers were at least 5-fold higher in the livers of LCMV-WE-infected mice than in the livers of LCMV-ARM-infected mice. Mice infected with the cytopathic viruses, mouse hepatitis virus and murine cytomegalovirus, experienced greater increases in NK/LGL by day 3 p.i. than did mice either infected with LCMV or injected with poly I:C. The early and late accumulations of LGL in the virus-infected liver were associated with the appearance of two waves of LGL with blast cell morphology expressing the phenotypes of NK cells and CTL, respectively. Thus, the organ-associated accumulation, blastogenesis, and in situ proliferation of cytotoxic LGL provide a means for the localization and site-specific augmentation of a host's cell-mediated antiviral defenses. The mechanism of inhibition of virus synthesis in vivo by immune splenocytes containing virus-specific CTL was examined in mice dually infected with two different viruses and then adoptively immunized with spleen cells immune to one of the two viruses. Only the titer of the virus to which the splenocytes were immune was reduced in titer, and no nonspecific antiviral effect was seen on the titer of the 'bystander' heterologous virus. These data are consistent with an in vivo mechanism of CTL-mediated antiviral resistance involving direct cytotoxicity rather than release and dissemination of antigen-nonspecific antiviral factors, such as interferon, following recognition of appropriate viral antigen.