Brain fatty acid-binding protein (B-FABP) is expressed in the radial glial cells of the developing central nervous system as well as in a subset of human malignant glioma cell lines. Most of the malignant glioma lines that express B-FABP also express GFAP, an intermediate filament protein found in mature astrocytes. We are studying the regulation of the B-FABP gene to determine the basis for its differential expression in malignant glioma lines. By DNase I footprinting, we have identified five DNA-binding sites located within 400 base pairs (bp) of the B-FABP transcription start site, including two nuclear factor I (NFI)-binding sites at -35 to -58 bp (footprint 1, fp1) and -237 to -260 bp (fp3), respectively. Competition experiments, supershift experiments with anti-NFI antibody, and methylation interference experiments all indicate that the factor binding to fp1 and fp3 is NFI. By site-directed mutagenesis of both NFI-binding sites, we show that the most proximal NFI site is essential for B-FABP promoter activity in transiently transfected malignant glioma cells. Different band shift patterns are observed with nuclear extracts from B-FABP(+) and B-FABP(-) malignant glioma lines, with the latter generating complexes that migrate more slowly than those obtained with B-FABP(+) extracts. All bands are converted to a faster migrating form with potato acid phosphatase treatment, indicating that NFI is differentially phosphorylated in B-FABP(+) and B-FABP(-) lines. Our results suggest that B-FABP expression in malignant glioma lines is determined by the extent of NFI phosphorylation which, in turn, is controlled by a phosphatase activity specific to B-FABP(+) lines.