Müllerian-inhibiting substance (MIS) is an essential factor for male sexual differentiation. In the present study, we examined whether the expression of MIS and several of its related transcription factors is altered in the ovaries of rats treated with the synthetic estrogen, E2 benzoate (EB; 10 microg/0.02 ml), from postnatal day 1 (P1) to P5. The EB-treated rats had a significantly reduced number of layered follicles at P6 in comparison with the control rats that were treated with vehicle alone. The expression levels of both MIS mRNA and protein in the granulosa cells of small growing follicles in the ovary at P6 were higher in the EB-treated rats than in the controls. These results indicate that the inhibitory effect of EB on the follicular stratification may correlate with the inappropriately increased expression level of MIS. Furthermore, the expression levels of one of its transcriptional activators, steroidogenic factor 1, and ER-beta in granulosa cells of small growing follicles were higher in EB-treated ovaries than in the control ovaries. These results suggest the role of MIS in the regulation of follicular growth and the possible involvement of steroidogenic factor 1 and/or ER-beta in this molecular cascade may contribute to postnatal ovarian development.