The intermediate filament protein nestin is expressed in the neural stem cells of the developing central nervous system (CNS). Promoter analysis revealed that the minimal promoter of the mouse nestin gene resides in the region )11 to +183 of the 5 0 -non-coding and upstream flanking region, and that two adjacent Sp1-binding sites are necessary for promoter activity. Electrophoretic mobility-shift assays (EMSA) and supershift assays showed that Sp1 and Sp3 proteins selectively bind to the upstream Sp1 site. These results demonstrate an important functionality of Sp1 and Sp3 in regulating the expression of the mouse nestin gene.