To assess prospectively the influence of the control of viral replication on the frequency of cytokine-producing T cells, and to correlate these changes with immune activation, we conducted a 15-month follow-up study of IFN-gamma- and IL-2-producing CD4+ and CD8+ T cells at a single-cell level in 12 previously untreated patients receiving highly active antiretroviral therapy (HAART). At baseline we observed a strikingly high proportion of IFN-gamma-producing CD8+ T cells. The treatment-induced decrease in the proportion of IFN-gamma-producing CD8+ T cells ran parallel to the decrease in HLA-DR+ and CD38+CD8+ T cell subsets and was associated with the reduction in HIV RNA level. IL-2-producing cells were mainly CD4+. As a consequence of CD4+ T cell loss, the number of IL-2-producing CD4+ T cells was lower in patients than in control subjects (52 vs. 171 cells/microl), but the proportion of these cells was unchanged (22.4 vs. 19.3). During therapy the proportion of CD4+ IL-2-producing cells was initially stable and then fell markedly at month 5, followed by a gradual return to previous values. The reduction in viral load was associated with the fall in the proportion of CD4+ activated subsets. Intracellular cytokine assays are a new approach to the assessment of T cell function in HIV infection. Our results suggest that the functional capacity of CD4+ T cells is probably less severely altered than previously thought on the basis of conventional assays. CD8+ T cells exhibit an increased capacity to produce IFN-gamma that is associated with an increase in activation marker expression. These alterations decrease partially and in parallel under treatment.