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The activity of virus-specific T lymphocytes, among which those capable of a polyfunctional response against the viral protein gag, is crucial to control HIV infection.The objective of this study is to investigate the polyfunctionality of gag-specific T cells in different phases of HIV infection, analyzing markers related to T-helper cell 1 (Th1) and degranulation/cytotoxicity, and the production of Th1 cytokines in peripheral blood lymphocytes from patients experiencing an acute primary infection, long-term nonprogressors, patients naive for antiretroviral drugs, and patients taking HAART.Cells were stimulated with a pool of gag-derived peptides or with a superantigen (staphylococcal enterotoxin B). Using eight-color polychromatic flow cytometry, we analyzed the expression of interleukin-2, interferon-γ, CD154, and CD107a by CD4+ and CD8+ T cells.The main finding was that in all HIV-positive patients, about half gag-specific CD4+ T cells were CD107a+, that is, able to degranulate. CD4+CD154+ cells unable to produce Th1 cytokines were the second most represented population. Truly polyfunctional CD4+ T cells were very rare and present only in a few long-term nonprogressors. Superantigen stimulation showed that CD4+ T lymphocytes from all patients displayed a typical Th response, including interleukin-2 and interferon-γ production, lacking CD107a expression.In all the aforementioned phases of HIV infection, the large majority of gag-specific CD4+ T lymphocytes cannot be identified by the sole expression of interleukin-2 and interferon-γ, which is early impaired. Degranulation and helper functions other than Th1 cytokine production are the predominant features of HIV-specific CD4+ lymphocytes.