Immunocompetent adult mice infected with lymphocytic choriomeningitis virus (LCMV) generate a strong antiviral cytotoxic T cell response that clears virus from all organs. Although there is good evidence that immune cytotoxic T lymphocytes (CTL) kill target cells in vitro, in vivo it is debated whether antiviral activity of CD8+ T cells is mediated via direct target cell lysis or via soluble mediators. To demonstrate CD8+ T cell-mediated destruction of infected cells in vivo a specific cell-internal releasable marker was used as label, i.e. the nucleoprotein (NP) of LCMV. Since LCMV is non-cytopathic the viral NP will only be released in substantial amounts because of destruction of infected host cells by immune CTL. It is shown here that the amount of NP released from infected and 51Cr-labeled target cells in vitro correlated well with the amount of radioactivity released. Viral NP released in vivo by CTL is bound and masked by the anti-NP antibodies that are produced very early and efficiently. However, NP could readily be detected in sera of LCMV-infected CD8+ competent mice that could not generate antibodies specific for the NP because they were treated with a depleting anti-CD4 antibody. NP was also detected in the cerebrospinal fluid of mice suffering from CD8+ T cell-mediated lymphocytic choriomeningitis after intracerebral infection. NP titers in sera of anti-CD4-treated LCMV-infected mice exhibited a peak around day 7-8 when CTL activity was highest. When mice were CD8 T cell-depleted with anti-CD8 monoclonal antibody or in LCMV-carrier mice, no NP was detected in the serum. Highly activated LCMV-specific CTL adoptively transfused to LCMV-infected irradiated recipient mice also caused a time-dependent release of NP into serum. This confirms that the CD8+ population is responsible for the release of NP from infected host cells. These results represent an in vivo correlate of CTL-mediated cytolysis and evidence that antiviral cytotoxic T cells are cytolytic in vivo. They also suggest that antibody responses to internal antigens of non-cytopathic viruses may signal CD8+ T cell-mediated destruction of infected host cells.