Glial fibrillary acidic protein (GFAP) is an intermediate filament protein present primarily in astrocytes. The gene is first expressed as astrocytes mature, and in the adult is strongly upregulated in response to CNS damage. Thus, in addition to its astrocyte specificity, transcriptional regulation of the GFAP gene is of interest as a reporter of CNS signaling during development and injury. Several laboratories have shown that approximately 2 kb of 5′-flanking DNA of the human or mouse GFAP gene is sufficient to direct transgene expression to astrocytes and to confer developmental and injury-induced regulation. Enhancer regions have been identified adjacent to the basal promoter and about 1500 bp upstream of the RNA start site. Juxtaposition of these two segments yielded a 447 bp promoter, gfa28, which strongly drove reporter activity in transfected glioma cells. We report here that in mice a gfa28-lacZ transgene expresses in only certain brain regions, revealing an unexpected heterogeneity among astrocytes. The restricted pattern of expression is present early in development, is not altered by injury, and is preserved in cultured astrocytes. However, astrocytes cultured from an inactive region strongly express a transiently transfected gfa28-lacZ construct, and activity of the embedded gfa28-lacZ transgene is partially restored by treatment with a histone deacetylase inhibitor. These results indicate that the absence of gfa28-lacZ expression in specific brain regions results from a developmental failure to remodel GFAP chromatin to an open structure. Thus, expression of the gfa28-lacZ transgene appears to serendipitously mark a distinct set of astrocyte precursors. © 2006 Wiley-Liss, Inc.