T cell clones are classified as type 0, 1 or 2 depending on the lymphokines they produce. However, it has remained unclear whether single cells of a given type produce one or several cytokine species. Flow cytometric analysis of peripheral blood lymphocytes (PBL) obtained from 20 healthy donors for the production of the type 1 cytokines IFN-+ and IL-2 revealed very few cells that co-expressed both cytokines independently of the mitogenic stimulus used for PBL activation. Similarly, kinetic studies of cytokine synthesis indicated a low percentage of IFN-+ /IL-2 double-positive T cells at all time points. Reverse transcription-PCR analysis of sorted IL-2-and IFN-+ -positive T cells showed the presence of IL-2-or IFN-+specific mRNA only in those cells expressing the corresponding cytokine. This segregation of the two type 1 cytokines was lost in long-term cultured T cells and in T cell clones. A high percentage of cells expressing only IL-2 or IFN-+ was observed even when the production of these cytokines was evaluated on CD4 + and CD8 + subsets. Moreover, in some healthy individuals, IFN-+ and IL-2 production by CD8 + T cells was related to CD8 + expression levels and cell size, i. e. IL-2-expressing cells were generally smaller with more intense CD8 + staining as compared with IFN-+ -producing T cells. These data indicate that activated T lymphocytes are strongly committed in vivo to produce IFN-+ or IL-2 and emphasizes the independent regulation of the two cytokine genes.