We previously reported cytotoxic activity of dengue virus-specific CD4+ CD8- T cell clones established in long-term in vitro culture. In the present experiments we tried to determine whether dengue virus-specific CD4+ CD8- CTL3 are present in short-term bulk cultures. Peripheral blood mononuclear cells (PBMC)3 from a donor who had been immunized with an experimental live attenuated dengue 1 vaccine 8 months earlier were used. PBMC were incubated with noninfectious dengue 1 antigen (Ag)3 for 7 days, and were examined for dengue 1-specific cytotoxic activity. PBMC cultured with dengue 1 Ag lysed autologous lymphoblastoid cell line (LCL)3 pulsed with noninfectious dengue 1 Ag, but did not lyse LCL pulsed with Ag of other dengue serotype, West Nile virus, or yellow fever virus, or control Ag. Treatment of cultured PBMC with monoclonal antibody to CD3 or CD4 and complement abrogated the cytotoxic activity but treatment with a monoclonal antibody to CD8 and complement did not. A time course study showed that dengue 1 Ag-specific CTL were first detected in 5 day cultures. Lysis of target cells by these CD4+ CTL were restricted by HLA class II, and HLA DQw1 and HLA DRw52 were determined to be the restriction molecules. These results indicate that dengue virus-specific CD4+ CD8- CTL are generated in short-term bulk cultures as well as in long-term-cultured cell lines, and support the concept that CD4+ CTL may be generated in vivo during infection.