We previously reported that the clone JK34 was cross-reactive for dengue virus types 1, 2, 3, and 4 and recognized NS3 (I. Kurane, M. A. Brinton, A. L. Samson, and F. A. Ennis, J. Virol. 65:1823-1828, 1991). In the present experiments, we defined the epitope at the amino acid level, with 93 15-mer overlapping peptides which cover the entire NS3. A peptide 4 which contains amino acids 251 to 265 of NS3 sensitized the autologous B lymphoblastoid cell line (LCL) to the lysis by JK34. The smallest peptide recognized by JK34 was a 10-mer peptide which contains amino acids 255 to 264 (EIVDLMCHAT). A monoclonal antibody to HLA-DP inhibited the lysis of epitope peptide-pulsed autologous LCL by JK34. Genotypic typing revealed that the HLA-DP of this donor is DPA1*01, DPB1*0201, which is serologically defined as HLA-DPw2. JK34 lysed peptide 4-pulsed allogeneic LCL which carried HLA-DPw2. These results indicate that HLA-DPw2 is the restriction allele for recognition of this epitope by JK34.