The role of dengue virus-specific serotype-cross-reactive T lymphocytes in recovery from and pathogenesis of dengue virus infections is not known. In the present paper, we have defined a dengue serotype-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK36 and JK46. These T cell clones were established from the peripheral blood T lymphocytes of a dengue-3-immune donor, using a limiting dilution method. JK36 and JK46 were cross-reactive for dengue virus types 2, 3, and 4, but not for type 1, and recognized the NS3 protein. The smallest synthetic peptide recognized by JK36 was an 8-amino acid peptide that contains amino acids (aa) 226 to 233 (VVAAEMEE) of NS3. The smallest peptide recognized by JK46 was an 11-amino acid peptide that contains aa 224 to 234 (TRVVAAEMEEA). HLA-DR15 was the restriction allele for recognition of these peptides by both JK36 and JK46. This is the first epitope to be defined that is recognized by human CD4+ CTL cross-reactive for dengue virus types 2, 3, and 4.