Using three-color flow cytometric analysis for the detection of intracellular cytokines, we have been able to determine the exact combination of cytokines produced by individual T lymphocytes. Because CD4+CD27- lymphocytes have been shown to produce more IL-4 and IL-5 than CD4+CD27+ lymphocytes, cells from normal individuals (n = 4) and helminth-infected patients (n = 4) were sorted magnetically for the CD4+CD27+ and the CD4+CD27- subpopulations. Intracellular staining for IL-4, IL-5, and IFN-gamma subsequent to mitogen stimulation for 6 h revealed that although almost no CD4+CD27- lymphocytes produce both IL-5 and IFN-gamma (0.03-1.4%), a distinct proportion produce both IL-4 and IFN-gamma (0.1-8.0%), and 66% to 84% of IL-5-producing cells also produce IL-4. Patients and normal individuals had the same functional T cell subsets, but the CD4+CD27- lymphocytes from patients had higher frequencies of cells producing IL-4 (geometric mean (GM), 24.3% vs 16.4%) or IL-5 (GM, 10.2% vs 2.9%), whereas those of normal individuals had higher frequencies of cells producing IFN-gamma (GM, 44.5% vs 17.2%; p = 0.043). These analyses also revealed that the CD4+CD27- population included significantly higher frequencies of cells that were IL-5+IFN-gamma- (GM, 4.9% vs 1.5%; p = 0.025), IL-4+IFN-gamma- (GM, 13.8% vs 3.5%; p = 0.025), and IFN-gamma+IL-4-IL-5- (GM, 27.3% vs 12.0%; p = 0.011) than the CD4+CD27+ population. Thus, we have clearly demonstrated Th1, Th2, and Th0 cell subsets within the CD4+-CD27- population of human lymphocytes.