The use of peptide–human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8⁺ T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8⁺ T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, and perforin is analyzed by FACS^® within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8⁺ T cells compared with cytomegalovirus (CMV)-specific CD8⁺ T cells in HIV chronic infection. We show that the majority of circulating CD8⁺ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α. However, a striking finding is that HIV-specific CD8⁺ T cells express significantly lower levels of perforin than CMV-specific CD8⁺ T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8⁺ T cells are impaired in cytolytic activity.