We used three-colour cytometry to analyse intracellular cytokine production in activated whole blood cultures derived from patients with HIV-1 infection. We assessed mitogen-induced IL-2, IL-4 and IFN-gamma production from T cells as possible markers of immune dysfunction. The percentages of T cells staining for IL-2 were significantly reduced in stimulated cultures from HIV+ individuals relative to normal controls (P<0.0001); this reduction was observed in both the CD4+ and the CD8+ subsets. IL-2 production was significantly reduced in CD4+ T cells from HIV+ individuals clinically classified as symptomatics compared with HIV+ asymptomatics (P<0.001); in addition, production of IL-2 inversely correlated with viral load (r2=0.832). On the other hand, HIV+ individuals showed significantly more T cells staining positive for IFN-gamma (P<0.0001); subset analysis identified these T cells as CD8+. Increased IFN-gamma production in the CD8+ T cell subset of HIV+ individuals correlated neither with clinical status nor with plasma viral load. IL-4 staining in activated T cells was low (<5%) and no differences were observed between HIV+ and control groups. Three-colour FACS analysis of whole blood provides a sensitive, rapid and relatively easy means to detect cytokine profiles within T cell subpopulations. Only small volumes of blood are required (0.5 ml), since there is no need for cell isolation, making it more practical than ELISA or reverse transcriptase-polymerase chain reaction (RT-PCR) for the analysis of immune function in HIV+ individuals. This technique could therefore play a role in mapping the dynamics and extent of immune recovery in AIDS patients undergoing triple combination therapy.