Glial fibrillary acidic protein (GFAP) is an intermediate filament specifically expressed in glial cells which helps to maintain and stabilize the glial cytoskeleton. Interestingly, with increasing astrocytic anaplasia, there is typically progressive loss of GFAP expression. In in vitro model systems, most permanent glioma cell lines are GFAP-negative. To determine the mechanism by which the transcription of the GFAP gene may be repressed in glioma cell lines, we initially performed a Southern analysis on a panel of human malignant glioma cell lines using a human cDNA probe for GFAP. By this method, no large rearrangements or deletions of the GFAP gene were found. Postulating that a change in methylation status of the GFAP gene could conceivably alter its expression in glioma cell lines, we studied the methylation state of the GFAP gene in the same glioma cell lines using a methyl-sensitive restriction enzyme digest of tumour and control DNA. Our analysis revealed that the GFAP gene was hypermethylated in 2/2 GFAP- negative glioma cell lines but not in 4/4 GFAP-positive glioma cell lines. To determine if methylation of CpG islands contained within the GFAP promoter could repress GFAP transcription, we designed deletional constructs from a 6 kb fragment of the mouse GFAP promoter, methylated them using Msp I- and Hpa II-methylases, and tested their activity in a standard CAT assay. Our data suggest that methylation of a 2 kb segment of the mouse GFAP promoter is sufficient to inactivate GFAP transcription. Our results further imply that methylation-mediated repression of GFAP transcription may be one candidate mechanism to account for decreased GFAP expression in certain human malignant glioma cell lines.