Daidzein and genistein are two prominent soy isoflavones that have been reported as promising protectors against cancers at many sites. In a study focusing on the chemopreventive mechanisms, we previously demonstrated that daidzein was an effective immune stimulator in an in vivo murine system. In this study we further evaluated the effects of daidzein and genistein, individually and in combination, on in vitro mitogen-stimulated activation of murine lymphocytes. At physiologically relevant concentrations (0.01-10.0 microM), daidzein significantly potentiated proliferation of mixed splenocyte cultures activated with concanavalin A or lipopolysaccharide in a dose-dependent manner in comparison with vehicle control, whereas genistein had no influence on the response. Although a significant cooperativity with genistein (1 microM) was observed at low concentrations of daidzein (0.01 microM) in comparison with daidzein alone, genistein failed to augment or counteract the effects of high concentrations of daidzein on lymphocyte proliferation. The secretion of cytokine interleukins-2 and -3 from concanavalin A-activated lymphocytes was significantly increased again by daidzein and was unaffected or mildly decreased by genistein. Taken together, these results demonstrate that daidzein, rather than genistein, is able to enhance in vitro activation of murine lymphocytes and suggest that more studies focusing on the immunoregulatory mechanism of soy daidzein and the potential clinical relevance are warranted.