Four brain-specific DNase I hypersensitive sites (HSS) have previously been identified flanking the mouse 68-kDa neurofilament gene within a 1.7 kb upstream sequence which confers neuronal specificity of expression of this gene in transgenic mice. Previously several DNA-binding factors were detected at the HSS closest to the transcription start site (HSS1). However, no major brain-specific factors were identified, suggesting a possible role for the three remaining HSS in conferring tissue-specificity to the NF-L gene. Sequence analysis of the NF-L promoter region demonstrated the presence of an extensive CT repeat and several potential binding sites which are also found in other neurofilament promoters. Gel mobility shift assays revealed a similar but not identical banding pattern with brain and liver nuclear extracts at HSS2, and HSS3, however the banding pattern for HSS4 was predominantly brain-specific. DNase I footprinting revealed several factors binding to the upstream HSS regions in brain and liver nuclear extracts. These include a CCAAT box at HSS2, a novel brain-specific footprint near an adenovirus promoter element E2aE-C beta and a single liver-specific footprint associated with an POU/octamer binding site at HSS4. The presence of brain-specific gel shift bands and tissue-specific footprints associated with HSS4, suggest that this region may play an important role in the regulation of the NF-L gene.