Outbred CD-1 mice were treated neonatally on Days 1–5 with the phytoestrogen, genistein (1, 10, or 100 μg per pup per day), and ovaries were collected on Days 5, 12, and 19. Ribonuclease protection assay analysis of ovarian mRNA showed that estrogen receptor β (ERβ) predominated over ERα in controls and increased with age. Genistein treatment did not alter ERβ expression, however, ERα expression was higher on Days 5 and 12. ERβ was immunolocalized in granulosa cells, whereas ERα was immunolocalized in interstitial and thecal cells. Genistein treatment caused a dramatic increase in ERα in granulosa cells. Genistein-treated ERβ knockout mice showed a similar induction of ERα, which is seen in CD-1 mice, suggesting that ERβ does not mediate this effect. Similar ERα induction in granulosa cells was seen in CD-1 mice treated with lavendustin A, a tyrosine kinase inhibitor that has no known estrogenic actions, which suggests that this property of genistein may be responsible. As a functional analysis, genistein-treated mice were superovulated and the number of oocytes was counted. A statistically significant increase in the number of ovulated oocytes was observed with the lowest dose, whereas a decrease was observed with the two higher doses. This increase in ovulatory capacity with the low dose coincided with higher ERα expression. Histological evaluations on Day 19 revealed a dose-related increase in multioocyte follicles (MOFs) in genistein-treated mice. Tyrosine kinase inhibition was apparently not responsible for MOFs because they were not present in mice that had been treated with lavendustin; however, ERβ must play a role, because mice lacking ERβ showed no MOFs. These data taken together demonstrate alterations in the ovary following neonatal exposure to genistein. Given that human infants are exposed to high levels of genistein in soy-based foods, this study indicates that the effects of such exposure on the developing reproductive tract warrant further investigation.