Abstract Glial fibrillary acidic protein (GFAP) is an intermediate filament protein which is primarily found in astrocytes. However, in eye diseases or when eye injuries occur, GFAP is expressed in large quantities in retinal Müller cells. The mechanism for this altered expression is unknown, but presumably involves injury-dependent signaling. The purpose of this study was to investigate regulation of GFAP gene expression in human Müller cells in vitro. Immunofluorescence, western blot, RT-PCR and Northern blot analyses were used to demonstrate the expression of GFAP in cultured Müller cells. Plasmids bearing various segments of the human GFAP promoter fused to a CAT reporter gene were used to transfect primary cultures of human Müller cells as well as the non-glial cell lines 293T and HeLa. Transcription directed by the GFAP promoter was found to be more than 50-times stronger in the Müller cells than in either of the non-glial cell lines, consistent with the data for endogenous GFAP expression. To our knowledge, this is the first study demonstrating GFAP gene regulation in human Müller cells. By examining the transcriptional activity of various segments from the human GFAP promoter, it can be concluded that the GFAP gene is differently regulated in Müller cells compared to glioma cell lines from the central nervous system (CNS).