Sex differentiation consists of multi-step pathway that involves expression of many different genes. Müllerian duct inhibitory substance (MIS) has a key role for regression of the Müllerian duct during male sex differentiation. Recently, endocrine disruptors (EDs), which often have estrogen-like activities, have caused concern over worldwide. It has been reported that estrogen regulates the MIS expression. Therefore, we tested whether ER alpha and ER beta influence the MIS promoter activity in the NT2/D1 cell line which expresses many sex differentiation-related genes such as SRY, SOX9, and DAX-1. RT-PCR analysis revealed that the NT2/D1 cells express both ER alpha and ER beta in addition to MIS. Under the low concentration of 17beta-estradiol (E2), the over-expression of exogenous ER alpha increased the MIS promoter activity 3.3-fold compared with the control. However, as E2 concentration was increased, the MIS promoter activity was decreased. For ER beta, we could not observe alterations of the MIS promoter activity. Furthermore, the over-expression of the exogenous SF-1 inhibited the activation of the MIS promoter with ER alpha. Although it remains unclear whether the effects of ER alpha on the MIS promoter are mediated through the genomic or the no-genomic actions, the present results suggest that ER alpha up-regulates the MIS promoter activity in the NT2/D1 cells under low concentrations of E2, and that the two ERs may work in different manners for the MIS promoter activation. The present findings may be useful to understand the molecular mechanisms by which EDs or estrogens affect the MIS expression.