Interleukin (IL)-4 and IL-5 are two cytokines which synergize in the induction of several biological effector functions. They are produced by mouse and human T helper 2 (Th2) and T helper 0 (Th0) cells. Little is known about the regulation of the two cytokines at the single-cell level. Here we show, using a flow cytometric intracellular staining technique, that IL-4 and IL-5 are predominantly produced by different human peripheral CD4+ and CD8+ T cells, whereas interferon (IFN)-gamma and IL-2 are produced by the same cells. In contrast, cloned human Th0 and Th2 cells were able to produce IL-4 and IL-5 simultaneously. The segregation of IL-4 and IL-5 in activated peripheral T cells was found within 72 h of activation upon anti-CD3 or phorbol ester + ionomycin stimulation. The kinetics of IL-4 and IL-5 production were different at the mRNA and the intra- and extracellular protein level, indicating that the cytokines are regulated differently. T cells from three patients with hyper-IgE syndrome did not display a substantial proportion of IL-4/IL-5 double-positive cells. However, simultaneous production could be induced in normal human T cells after prolonged stimulation with a minimum of two restimulation cycles. We conclude that the simultaneous production of IL-4 and IL-5 is a feature of repetitively activated human T cells.