AbstractDietary phytoestrogens have been implicated in infertility among ruminants and may relate to human breast cancer risk. Formononetin is an isoflavonoid phytoestrogen found in animal fodder and in certain human foodstuffs. To investigate a possible mechanism by which phytoestrogens might influence mammary carcinogenesis, this study examined the capacity of formononetin to stimulate mammary gland proliferation. Formononetin was administered to castrated female BALBIc mice by daily subcutaneous injection; then mammary gland proliferation and estrogen receptor expression were quantified, and plasma prolactin levels were measured. A preliminary dose‐finding study demonstrated an estrogenic effect on vaginal cytology when formononetin was injected at 40 mg/kg sc. Peak plasma concentrations of 2.5±0.8 (SD)μg/ml at two hours and peak mammary tissue concentrations of 2.0±0.2 ng/mg tissue at four hours were noted after a single injection at this minimally bioactive dose. Among animals treated with formononetin at 40 mg/kg/day for five days, mammary gland proliferation was enhanced 3.3‐fold over saline‐treated controls and was comparable to that of animals treated with estradiol‐17βat 1μg/kg/day for five days. Mammary tissue estrogen receptor expression was 2‐fold higher among the formononetin‐treated animals (P<0.01 vs. saline‐treated controls), and plasma prolactin concentrations were increased 1.7‐fold (P<0.001 vs. saline‐treated controls). In subsequentin vitrobinding studies, formononetin competitively bound murine mammary estrogen receptors, but with a relative binding affinity 15,000 times less potent than that of estradiol‐17β. The results demonstrate an ability of formononetin to support mammary gland proliferation. However, the estrogenic potency of formononetin appears extremely weak compared with that of estradiol‐17βand is roughly proportional to estrogen receptor‐binding capacity.