The functional status of circulating human immunodeficiency (HIV)-specific CD8 T cells in chronically infected subjects was evaluated. By flow cytometry, only 5 of 7 subjects had detectable CD8 T cells that produced IFN-γ after stimulation with HIV-infected primary CD4 T cells. In 2 subjects, the frequency of IFN-γ–producing cells increased 4-fold when IL-2 was added to the culture medium; in another subject, IFN-γ–producing cells could be detected only after IL-2 was added. IFN-γ–producing cells ranged from 0.4% to 3% of CD8 T cells. Major histocompatibility complex–peptide tetramer staining, which identifies antigen-specific T cells irrespective of function, was used to evaluate the proportion of HIV-specific CD8 T cells that may be nonfunctional in vivo. CD8 T cells binding to tetramers complexed to HIV gag epitope SLYNTVATL and reverse transcriptase epitope YTAFTIPSI were identified in 9 of 15 and 5 of 12 HLA-A2–expressing seropositive subjects at frequencies of 0.1% to 1.1% and 0.1 to 0.7%, respectively. Freshly isolated tetramer-positive cells expressed a mixed pattern of memory and effector markers. On average, IFN-γ was produced by less than 25% of tetramer-positive CD8 T cells after stimulation with the relevant gag or reverse transcriptase peptide. In all subjects tested, freshly isolated CD8 T cells were not cytolytic against peptide-pulsed B lymphoblastoid cell line or primary HIV-infected CD4 T-cell targets. Exposure to IL-2 enhanced the cytotoxicity of CD8 T cells against primary HIV-infected CD4 targets in 2 of 2 subjects tested. These results suggest that a significant proportion of HIV-specific CD8 T cells may be functionally compromised in vivo and that some function can be restored by exposure to IL-2.