Progressive HIV disease has been associated with loss of memory T cell responses to Ag. To better characterize and quantify long-lived memory T cells in vivo, we have refined an in vivo labeling technique to study the kinetics of phenotypically distinct, low-frequency CD8+ T cell subpopulations in humans. HIV-negative subjects and antiretroviral-untreated HIV-infected subjects in varying stages of HIV disease were studied. After labeling the DNA of dividing cells with deuterated water (2 H 2 O), 2 H-label incorporation and die-away kinetics were quantified using a highly sensitive FACS/mass spectrometric method. Two different populations of long-lived memory CD8+ T cells were identified in HIV-negative subjects: CD8+ CD45RA− CCR7+ CD28+ central memory (T CM) cells expressing IL-7Rα and CD8+ CD45RA+ CCR7− CD28− RA effector memory (T EMRA) cells expressing CD57. In pilot studies in HIV-infected subjects, T CM cells appeared to have a shorter half-life and reduced abundance, particularly in those with high viral loads; T EMRA cells, by contrast, retained a long half-life and accumulated in the face of progressive HIV disease. These data are consistent with the hypothesis that IL-7Rα+ T CM cells represent true memory CD8+ T cells, the loss of which may be responsible in part for the progressive loss of T cell memory function during progressive HIV infection.