Nestin is a characteristic intermediate filament protein expressed in neural stem cells (NSCs). Evidence has shown that it is also found in reactive astrocytes. Previous studies have demonstrated that the second intron of the human and rat nestin genes harbors the central nervous system (CNS) enhancer and the midbrain enhancer, which regulate nestin expression in different regions of CNS during development. In this study, using an NSC-like cell line C17.2 and primarily cultured astrocytes, we show that both C17.2 cells and astrocytes express nestin. To characterize the nestin enhancer in further detail, we cloned the second intron of the mouse nestin gene, which is homologous to the human and rat counterparts as shown by DNA sequencing. Reporter assay indicated that the full-length nestin enhancer was active in both C17.2 cells and astrocytes, consistent with the immunocytochemistry results. However, in C17.2 cells, the enhancer activity was attributed to the highly conserved 3' part, and the 5' part of the enhancer was suppressive to the transcription activation activity of the full-length enhancer. While in astrocytes, both 3' and 5' parts were able to enhance the reporter gene expression. Our data suggested that different regions of the nestin enhancer might have different functions in C17.2 cells and astrocytes: while the 3' region activates transcription in both cell types, the 5' region suppresses in C17.2 cells but activates in astrocytes nestin expression.