Myelin-associated glycoprotein (MAG) is emerging as an important molecule involved in the plasticity and regeneration of the central nervous system. In this study, the structure of MAG gene promoter was characterized in cultured rat oligodendrocyte lineage cells. Heterogeneous transcription initiation with five major and eight minor start sites scattered within 72 bp was shown by primer extension analysis. This TATA-less core promoter contains no prominent initiator (Inr) elements associated with the transcription initiation sites, and hence, appears to utilize novel positioning mechanisms. Genomic footprinting analysis revealed several putative protein-binding regions overlapping the initiation sites and containing a multitude of CG-rich sequences. However, no conspicuous alterations in the protein-binding pattern were evident between O2A progenitors in which the gene is inactive, and mature oligodendrocytes with fully upregulated gene. The core promoter DNA features a differentiation-dependent demethylation as shown by genomic sequencing analysis. Three of eight cytosines are totally demethylated in oligodendrocyte chromosomes, indicating that these unmodified bases may be critical for full activation of the promoter. The core promoter is located within an internucleosomal linker, and the upstream regulatory region appears to be organized into an array of nucleosomes with hypersensitive linkers.