In the mammalian central nervous system (CNS), multipotential neural stem cells in the developing neuroepithelium generate the three major types of neural cells, namely neurons, astrocytes, and oligodendrocytes.To explore the molecular mechanisms controlling proliferation and differentiation of the neural stem cells, we have established clonal cell lines (named MNS cell lines) from the embryonic day 11-12 rat neuroepithelium. The MNS cell lines were immortalized by introducing the mycer fusion gene, whereby the c-myc is fused to the ligand-binding domain of the estrogen receptor, which enables conditional activation of the c-myc protein by adding estrogen (e.g., b-E2) in the culture medium. The MNS cells express nestin, vimentin, and the RC1 antigen, which are the markers of undifferentiated neuroepithelial cells. Furthermore, the MNS cell lines can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they possess the properties of multipotential neural stem cells. By using these cell lines we analyzed the expression of various types of genes that are implicated for neural development. RT-PCR and Northern blot analyses demonstrated that all of our cell lines examined expressed many potentially important cell fate regulatory genes, including MASH-1, E47, HES-1, HES-5, Id1, Notch1, and Notch2. On the other hand, several genes that have discrete expression domains in the developing brain and postulated to be involved in regional specification of the CNS (Pax-3, Pax-5, Pax-6, Dlx-1, Dbx, Otx1, Emx2, and Wnt-3) were differentially expressed in distinct cell lines. Furthermore, a characteristic profile of regulation of these genes was observed in association with the proliferation and differentiation of each cell line. These results suggest the intriguing possibility that the MNS cell lines bear basic characteristics of multipotential neural stem cells, and at the same time inherit the positional identity of the single neuroepithelial cells from which the cell lines were derived.Based on these observations, possible use of our cell lines to study the molecular mechanisms that control the generation of neural cell diversity in the developing mammalian CNS will be discussed.