The gfa gene encodes glial fibrillary acidic protein, an intermediate filament protein found almost exclusively in astrocytes. Transient transfection studies with a chloramphenicol acetyltransferase reporter gene were used to identify regions of the gfa gene responsible for its expression. Three regions, A, B, and D, were found to be important. The D region is located near the basal promoter, while A and B are next to each other about 1500 bp further upstream. The regions contain several sequences homologous to binding sites of known transcription factors, and in addition, each contains an identical novel 10-bp motif. The A, B, and D regions act in a cell-specific manner; when joined to the SV 40 early promoter, they enhance transcription in the glial cell line U251, but not in the nonglial cell line HepG2. Consistent with this observation, the DNase I footprint produced in these regions by nuclear extract from U251 cells differs from that produced by an extract from HepG2 cells. The B region appears to be the most active of the three, as by itself it stimulates strong cell-specific transcription, whereas addition of the other two regions has little effect. When the B region is at its normal distance from the basal promoter, deletion of D severely reduces transcription, but when B is placed near the promoter, D is unimportant. This suggests that the D region may function primarily to promote interactions that bring B close to the promoter.