Isoflavones are known to exert anticancer effects. These effects were examined using two isoflavones, biochanin A and daidzein, in a mouse mammary tumor virus (MMTV)-induced spontaneous breast cancer model. Emphasis was placed on isoflavone metabolism by the intestinal microflora and changes in estrogen levels. Germ-free (Gf) mice and their conventionalized (Cv) counterparts were assigned to three diet groups: (1) control diet, (2) biochanin A and (3) daidzein. In all groups, urine was collected from virgin female mice to analyze isoflavone metabolism by high performance liquid chromatography. These studies revealed changes of biochanin A into genistein, and of daidzein into equol, which were accelerated in the Cv animals. However, the Gf mice could not transform biochanin A into genistein, or daidzein into equol. Estrogen levels in the control and daidzein diet groups were lower in the Gf mice than in the Cv mice. The biochanin A group showed no differences in estrogen levels between the Cv and Gf animals. Four-week-old male and female animals were paired in the Gf and Cv groups. The female animals delivered and lactated repeatedly and were observed for the development of mammary cancer by palpation, twice weekly, until 15 months of age. The Cv mice showed a significantly lower incidence of breast cancer in the biochanin A diet group than in the control or daidzein groups (p < 0.05). These results suggest that the anticarcinogenic effects in this system might be produced not by daidzein or equol, but by biochanin A and/or genistein. In the Gf animals, the incidence of breast cancer was significantly higher in the biochanin A group than in the control group (p < 0.05), probably due to the increased level of estradiol in the former group. The biochanin A group tended to have a higher incidence of breast cancer than the daidzein group in the Gf group, although no significant differences were noted. Thus, no anticarcinogenic effect was produced by biochanin A alone in the Gf mice. In view of the results presented, genistein derived from biochanin A following metabolic processes in the intestinal microflora most likely acts as an inhibitor in breast carcinogenesis; biochanin A is most likely a precursor of genistein.