The mechanisms that underlie Th1/Th2 differentiation of human T cells are incompletely defined. In the present study, a panel of human T-cell clones was used to elucidate the relationship between Th1/Th2-specific transcription factors and cytokine production in human helper T cells. The mRNA expression level of T-bet, a Th1-specific transcription factor, was higher in Th1 clones than in Th2 clones. In contrast, inducible expression of Th2-specific transcription factors (GATA-3 and c-Maf) in Th2 clones was higher than that in Th1 clones. The expression level of T-bet in various T-cell clones was positively correlated with that of IFN-gamma and negatively correlated with that of Th2 cytokines, particularly IL-4. Interestingly, the expression of IL-3 and IL-13, but not of other Th2 cytokines IL-4 and IL-5, was strongly correlated with GATA-3 mRNA levels. A reduction of GATA-3 using RNA interference technology suppressed, whereas overexpression of GATA-3 enhanced, the expression of IL-3 and IL-13. In conclusion, the level of T-bet expression is correlated with Th1/Th2 polarization status, whereas GATA-3 is a crucial factor in determining the IL-3 and IL-13 producing capacity of human T cells.