Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)‐2 production in vitro, but its role in IL‐4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti‐CD3, phorbol 12‐myristate 13‐acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti‐CD28 in repetitive stimulation‐rest cycles. Initial stimulation of CD4⁺ cells with anti‐CD3 (or the mitogens PHA or PMA+ionomycin) and anti‐CD28 monoclonal antibodies induced IL‐4, IL‐5 and interferon‐γ (IFN‐γ) production and augmented IL‐2 production (6‐ to 11‐fold) compared to cells stimulated with anti‐CD3 or mitogen alone. The anti‐CD28‐induced cytokine production corresponded with augmented IL‐4 and IL‐5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL‐4 and IL‐5 production and diminished IL‐2 and IFN‐γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2‐like response correlated with an increased frequency of IL‐4‐secreting cells; up to 70% of the cells produced IL‐4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2‐type cells. This CD28‐mediated response was IL‐4 independent, since enhanced IL‐5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti‐IL‐4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.