We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h Cr-51-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alphabeta and TCR gammadelta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gammadelta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, >98% pure alphabeta and gammadelta T cells, it was found that alphabeta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific Cr-51-release, while gammadelta T cells showed only 2-4% Cr-51-release. These data indicate that REV-specific CTL response is mediated by alphabeta T cells and that gammadelta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.