Neural stem cells generate a wide spectrum of cell types in developing and adult nervous systems. These cells are marked by expression of the intermediate filament nestin. We used the regulatory elements of the nestin gene to generate transgenic mice in which neural stem cells of the embryonic and adult brain are marked by the expression of green fluorescent protein (GFP). We used these animals as a reporter line for studying neural stem and progenitor cells in the developing and adult nervous systems. In these nestin-GFP animals, we found that GFP-positive cells reflect the distribution of nestin-positive cells and accurately mark the neurogenic areas of the adult brain. Nestin-GFP cells can be isolated with high purity by using fluorescent-activated cell sorting and can generate multipotential neurospheres. In the adult brain, nestin-GFP cells are approximately 1,400-fold more efficient in generating neurospheres than are GFP-negative cells and, despite their small number, give rise to 70 times more neurospheres than does the GFP-negative population. We characterized the expression of a panel of differentiation markers in GFP-positive cells in the nestin-GFP transgenics and found that these cells can be divided into two groups based on the strength of their GFP signal: GFP-bright cells express glial fibrillary acidic protein (GFAP) but not betaIII-tubulin, whereas GFP-dim cells express betaIII-tubulin but not GFAP. These two classes of cells represent distinct classes of neuronal precursors in the adult mammalian brain, and may reflect different stages of neuronal differentiation. We also found unusual features of nestin-GFP-positive cells in the subgranular cell layer of the dentate gyrus. Together, our results indicate that GFP-positive cells in our transgenic animals accurately represent neural stem and progenitor cells and suggest that these nestin-GFP-expressing cells encompass the majority of the neural stem cells in the adult brain.