Natural killer (NK) cells can directly recognize virus-infected cells. Here, we demonstrate that NK cells also produce interferon (IFN)-gamma in an HIV-1-specific, T cell-dependent manner. After stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-infected individuals with HIV-1-derived peptides, up to half of the IFN-gamma-producing PBMCs are NK cells. These results indicate that T cell-dependent NK cell IFN-gamma production can be important for immune control of HIV-1, and have implications for the interpretation of data from vaccine trials using ELISPOT and ELISA.