Genistein is a phytoestrogen contained at high levels in soy products and has been shown to regulate immunoresponse. In this study, we evaluated the effects of genistein on the production of cytokines from antigen (Ag)-specific T cells using DO11.10 transgenic mice because the direct effect of genistein on Ag-specific cytokine production has not been elucidated. The oral administration of 20 mg/kg genistein increased IFN-gamma and IL-4 production from DO11.10+ T cells in response to ovalbumin (OVA)323-339 peptide in female DO11.10 mice. Analysis of intracellular cytokine synthesis revealed that the percentages of cytokine-producing cells in the control and genistein-treated groups were not different, indicating that increased cytokine production occurred at the single-cell level. In contrast to the female mice, genistein did not increase cytokine production in male mice, suggesting that the effect of genistein on cytokine production is gender-dependent.