Use of soy-based infant formulas and soy/isoflavone supplements has aroused concern because of potential estrogenic effects of the soy isoflavones genistein and daidzein. Here we show that s.c. genistein injections in ovariectomized adult mice produced dose-responsive decreases in thymic weight of up to 80%. Genistein's thymic effects occurred through both estrogen receptor (ER) and non-ER-mediated mechanisms, as the genistein effects on thymus were only partially blocked by the ER antagonist ICI 182,780. Genistein decreased thymocyte numbers up to 86% and doubled apoptosis, indicating that the mechanism of the genistein effect on loss of thymocytes is caused in part by increased apoptosis. Genistein injection caused decreases in relative percentages of thymic CD4⁺CD8 and double-positive CD4⁺CD8⁺ thymocytes, providing evidence that genistein may affect early thymocyte maturation and the maturation of the CD4⁺CD8 helper T cell lineage. Decreases in the relative percentages of CD4⁺CD8 thymocytes were accompanied by decreases in relative percentages of splenic CD4⁺CD8 cells and a systemic lymphocytopenia. In addition, genistein produced suppression of humoral immunity. Genistein injected at 8 mg/kg per day produced serum genistein levels comparable to those reported in soy-fed human infants, and this dose caused significant thymic and immune changes in mice. Critically, dietary genistein at concentrations that produced serum genistein levels substantially less than those in soy-fed infants produced marked thymic atrophy. These results raise the possibility that serum genistein concentrations found in soy-fed infants may be capable of producing thymic and immune abnormalities, as suggested by previous reports of immune impairments in soy-fed human infants.