Stimulation of human vaccinia virus immune peripheral blood mononuclear cells in vitro from vaccinia virus-immune donors with live vaccinia virus-infected autologous cells generated vaccinia virus-specific cytotoxic T lymphocytes (CTL) capable of lysing vaccinia virus-infected cells. We generated vaccinia virus-specific CD8+ clones and CD4+ CTL lines by limiting dilution from two donors by using peripheral blood mononuclear cells obtained 2 months or 4 years postrevaccination with vaccinia virus. These results demonstrate that vaccinia virus-specific CTL are generated as a result of immunization of humans with vaccinia virus and that both CD8(+)- and CD4(+)-specific T cells are maintained as memory cells.