SUMMARY We describe the highly conserved sequence 56–68 of the HIV Nef protein as the first promiscuous HLA-DQ HIV-derived peptide. The Nef peptide exhibits an albeit rare capacity to bind 6 different HLA-DQ molecules whereas no binding is observed with the 10 HLA-DR molecules tested. In agreement with these data, after immunization with the Nef peptide, HLA-DQ transgenic Aβ° mice display a vigorous cellular and humoral response while the specific immune response of HLA-DR expressing mice is minimal. The promiscuous potentiality of the Nef 56–68 peptide in humans has been confirmed by ex vivo immunization experiments with CD4+ T cells from 14 healthy donors expressing different HLA genotypes. Nef 56–68 specific CD4+ T cells rapidly acquire a memory cell phenotype and are characterized by the preferential usage of the TCR Vβ 6·1 gene segment and predominant production of IFN-γ. Taken together, these data indicate that the Nef 56–68 peptide constitutes an attractive component of vaccines aiming at inducing or enhancing HIV-specific T cell immunity.