Glial fibrillary acidic protein (GFAP), an intermediate filament protein normally found in astrocytes, and the radial glial marker brain fatty acid-binding protein (B-FABP; also known as FABP7) are co-expressed in malignant glioma cell lines and tumors. Nuclear factor I (NFI) recognition sites have been identified in the B-FABP and GFAP promoters, and transcription of both genes is believed to be regulated by NFI. Here, we study the role of the different members of the NFI family in regulating endogenous and ectopic B-FABP and GFAP gene transcription in human malignant glioma cells. We show by gel shifts that all four members of the NFI family (NFIA, NFIB, NFIC, and NFIX) bind to B-FABP and GFAP NFI consensus sites. Over-expression of NFIs, in conjunction with mutation analysis of NFI consensus sites using a reporter gene assay, supports a role for all four NFIs in the regulation of the GFAP and B-FABP genes. Knock-down of single or combined NFIs reveals promoter-dependent and promoter-context-dependent interaction patterns and suggests cross talk between the different members of the NFI family. Our data indicate that the NFI family of transcription factors plays a key role in the regulation of both the B-FABP and GFAP genes in malignant glioma cells.