We tested two biological properties of a continuously growing mouse cytotoxic T cell line, L4, which is specific for influenza A virus and has been cloned and recloned many times. We previously reported that L4 cells are H-2 restricted and cross-reactive for all type A influenza viruses, whereas they do not recognize type B influenza viruses. They bear Thy-1 and Lyt-2 markers. In the present study, we show that L4 cytotoxic T cells protect mice against a lethal influenza infection on transfer to syngeneic recipients, and reduce virus titers in the lungs of mice challenged with a heterologous type A influenza virus. This provides further support for the active role of cytotoxic T cells in limiting virus replication in influenza infection. We could also demonstrate that the cloned cytotoxic T cells induce a delayed-type hypersensitivity skin reaction in the footpads of mice challenged with live or inactivated influenza virus. This reaction can be observed at 24 h, but has declined by 48 h. A clone of cells derived from L4 that has lost its cytotoxic potential and its ability to recognize infected cells did not induce a delayed-type hypersensitivity reaction in the presence of virus. Thus, cytotoxic T cells actively killing influenza virus-infected cells are able to induce a delayed-type hypersensitivity skin reaction to homologous and heterologous type A influenza viruses.