We examined nine dengue virus-specific human CD4+ CD8- cytotoxic T lymphocyte (CTL) clones for protein recognition, using recombinant vaccinia viruses which contain genes coding for dengue virus proteins. These clones were established from peripheral blood mononuclear cells of a donor previously immunized with a live-attenuated experimental dengue-1 vaccine. Of nine CD4+ T cell clones, seven were dengue-1-specific and two were dengue-1-dengue-3 cross-reactive. Four dengue-1-specific clones and one dengue-1-dengue-3 cross-reactive clone recognized epitopes within the NS1 or NS2a proteins. Analysis of human leukocyte antigen (HLA) restriction revealed that three dengue-1-specific clones are HLA-DR1-restricted and one dengue-1-dengue-3 cross-reactive clone is HLA-DPw3-restricted. These results indicate that NS1 and NS2a proteins as well as C, E, and NS3 proteins reported earlier contain one or more epitopes recognized by dengue virus-specific human CD4+ T lymphocytes.