Natural and synthetic estrogens, Including diethylstilbestrol (DES), given during the critical period of newborn life Induce abnormalities in ovaries of mice. Induction of polyovular follicles (PFs) containing two or more oocytes in a follicle is one example. In this study, the involvement of estrogen receptor subtypes ERα and ERβ in induction of PFs by neonatal treatment with DES was analyzed by using ERα knockout (αERKO) and ERβ knockout (βERKO) mice. Ovaries of mice injected with 3 µg DES for 5 days from the day of birth were examined histologically from 10 to 60 days of age, and the expression of genes involved in folliculogenesis was analyzed by real-time quantitative PCR. The PF incidence (percent of PFs per 100 follicles greater than 50 µm in diameter) in the ovary of αERKO mice treated with DES was not different from that in the DES-treated wild-type mice. However, neonatal DES treatment did not increase the PF incidence in βERKO mice, suggesting that PFs were induced by DES through ERβ but not ERα. The expression of bone morphogenetic protein 15, growth differentiation factor 9, inhibin-α, Müllerian inhibiting substance, and other genes in the ovaries of DES-treated βERKO mice was not different from that in the ovaries of DES-treated wild-type mice. These results indicate that ERβ but not ERα is essential for DES to induce PFs in mice.