Double negative (DN) T cells are CD3(+), CD4(-), CD8(-) cells with either T-cell receptors (TCR) αβ or TCR γδ whose importance on protection against HIV infection is unknown. Since HIV-exposed seronegative individuals correspond to an ideal group in whom correlates of protection are expected, the role of these cells was studied in 13 HIV-serodiscordant couples in a stable relationship and reporting unprotected sexual intercourses. HIV-specific immune responses mediated by DN T-cells were evaluated by measuring intracellular IFNγ and MIP1β (CCL4) production in response to HIV-Gag peptides. Thirty-five healthy controls not exposed to HIV were tested similarly and used to define a threshold for positive responses. Interestingly, Gag-specific DN T-cell responses were found in 3/13 (23%) HIV-exposed seronegative individuals (Group A), involving both DN/αβ(+) and DN/γδ(+) T-cells through MIP1β and IFNγ production. 4/13 (30%) of partners infected with HIV (Group B) also showed Gag-specific responses but were mediated exclusively by DN/γδ(+) T-cells, mainly through IFNγ production. DN T-cells in Group A individuals can display differential HIV-specific immune responses, which might contribute to the low susceptibility to infection with HIV shown by individuals in Group A.