A diet high in soy is associated with many health benefits, including reduced incidence of breast cancer. The soy phytoestrogen, genistein, is hypothesized to contribute to mammary chemoprevention via interaction with estrogen receptors (ERs) alpha and/or beta. These steroid signaling pathways are believed to exert control over proliferation and differentiation of the mammary gland by a complex bidirectional interaction with the epidermal growth factor (EGF) signaling pathway. The current work was designed to study the role of these two pathways in prepubertal mammary gland growth. Female Sprague-Dawley CD rats were injected with genistein (500 μg/g body wt) or estradiol benzoate (EB) (500 μg/g body wt) on days 16, 18 and 20. Whole mount analysis of mammary glands from 21-day-old rats showed that both treatments resulted iif significantly increased terminal end buds (TEBs), and increased ductal branching, compared with animals given the vehicle, dimethylsulfoxide (DMSO). Both effects were inhibited by blockage of ER function by pre-treating with 2 mg ICI 182,780/kg body wt, a steroidal anti-estrogen. Immunoblotting analyis of mammary gland extracts demonstrated increased epidermal growth factor receptor (EGFR) and progesterone receptor (PR) expression following treatment with EB or genistein. Tyrosine-phosphorylated EGFR, as measured by immunoprecipitation/immunoblotting was also increased, but when normalized to total receptors, there was no net effect. The expression and phosphorylation of downstream targets of the EGFR, mitogen activating kinase kinase (MEK 1 and 2) and extracellular signal regulated kinases 1 and 2 (ERK 1 and 2) were not significantly affected. Anti-estrogen pre-treatment prevented the increase in EGFR, phospho-EGFR and PR. The data indicate an ER-based mechanism of action for genistein in mammary gland proliferation and differentiation, which can lead to protection against mammary cancer.