Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein in astrocytes, one of the most abundant cell types in the vertebrate central nervous system (CNS). Transcriptional regulation of GFAP is of interest because of its astrocyte-specificity and its upregulation during development and CNS injury. A 2.2 kb human GFAP promoter, gfa2, has been found to express in astrocytes throughout the CNS. In contrast, we recently found that the 448 bp gfa28 promoter expresses in only restricted CNS regions, and is active in neurons as well as astrocytes. In the present study we have used transgenic mice to investigate which DNA regions deleted from gfa2 in the formation of gfa28 are responsible for these differences. We have found that a 55 bp segment spanning bp -1488 to -1434 with respect to the RNA start site contains region-specific elements and that a 45 bp sequence spanning bp -1443 to -1399 is required for silencing expression in neurons. These data also further confirm the heterogeneity of astrocytes and neurons in the activation and repression of the GFAP gene, respectively. These studies also generated a novel 681 bp GFAP promoter, gfaABC1D, that has essentially the same expression pattern as the 2210 bp gfa2 promoter, and about twofold greater activity, recommending it for gene targeting applications in which size matters. In addition, a 681 bp gfaABC1(mC1.1)D variant was generated that could limit expression of transgenes to astrocytes in the dorsal and caudal cortex, hippocampus and caudal vermis of the cerebellum. © 2008 Wiley-Liss, Inc.