Cytotoxic T lymphocytes were functionally demonstrated in spleen cells from chickens 7 days post inoculation with reticuloendotheliosis virus using a Cr-release assay. Major histocompatibility complex (MHC)-restricted cytotoxicity was demonstrated using effector and target cells from two different strains of chickens of known avian MHC haplotype. Anti-viral specificity was shown and in vivo generation of MHC-restricted cytotoxicity was evaluated. Cytotoxic T cells were distinguished from macrophages and natural killer cells. Their cytotoxicity was not antibody dependent. Higher levels of cytolysis were found with cytotoxic T cells from embryonally bursectomized vs. intact chickens over a large range of effector to target cell ratios. Using monoclonal antibodies, cytotoxic T cells were further defined as Ia+ T cells by immunofluorescence, antibody plus complement-mediated lysis of effector cells and blocking of cytolysis in the Cr-release assay.