Isoflavone (ISO) exposure during adolescence has been demonstrated to modulate the estrogenic and proliferative sensitivity of the adult breast tissue. In this study, we investigated whether ISO exposure restricted to the period of puberty is sufficient to result in similar effects. Female rats were divided into three groups receiving either lifelong an ISO-free diet (IDD) or an ISO-rich diet (ISD), or an ISD from postnatal day (PND) 30 to PND 60 covering the time period of puberty (pISD). Serum concentrations of ISO and metabolites were determined at PND 50 and 80. At PND 50, ISD animals had significant higher equol serum levels than pISD animals. Onset of puberty occurred significantly earlier in the pISD and ISD group compared to the animals fed IDD. Cycle length was shortest in pISD group. To determine estrogen sensitivity of the adult breast tissue, adult rats were ovariectomized and subcutaneously treated either with estradiol (E(2)) or with genistein (GEN) for 3 days (PND 77-80). Analysis of Ki-67 and proliferating cell nuclear antigen (PCNA) expression showed a reduced proliferative response of the breast to E(2) in pISD and ISD animals compared to IDD group, while the induction of progesterone receptor (PR) was higher in both IDD and pISD compared to ISD fed rats. Our results demonstrate that ISO exposure during puberty is sufficient to reduce the proliferative response of the adult mammary gland but not to reduce the response of classical E(2) sensitive genes like the PR. In summary, our results demonstrate that animals exposed during different periods of their adolescence to ISO differ in several physiological aspects. In addition, the detected differences in the serum equol levels between pISD and ISD rats and the detected differences in the estrogen sensitivity of the breast clearly underline this assumption.