The methylation status of a 4.4-kb 5' end of the myelin-associated glycoprotein (MAG) gene was assessed in cells with different levels of transcriptional activity of the gene, i.e., liver, brain, O-2A oligodendrocyte precursors cells, mature oligodendrocytes, and glioma C6 cells. Purified DNA was digested with methylation-sensitive restriction enzymes, and the cuts were mapped by the indirect end-labeling technique. The restriction sites within the 4.4-kb fragment revealed a highly heterogenous methylation pattern among cells and tissues, and liver DNA was the most heavily methylated. Most of the restriction sites were partly demethylated in the nervous system cells. Notably, two adjacent Hha1 sites at +94 and +96 were fully methylated in liver, but partially demethylated in the brain, OL, and O2A. Two Hpa2 site located at -1836 and at -39 were progressively demethylated in oligodendrocyte lineage cells, indicating specific hypomethylation associated with the oligodendrocytic differentiation. Most of the restriction sites were weakly methylated in the DNA from neoplastic C6 cells, although the Hha1 sites were fully methylated. No clear-cut correlation between the extent of CpG dinucleotide methylation and the chromatin conformation was found. For example, out of four heavily methylated sites only two comapped with MNase hypersensitive sites. Also, the -1836 Hpa2 site whose demethylation is concomitant with oligodendrocytic differentiation seems to be localized within precisely positioned nucleosomal arrays of the MAG gene chromatin. The results indicate that the MAG gene undergoes progressive demethylation concomitant with the oligodendrocyte differentiation/maturation. However, certain CpG dinucleotides remain heavily methylated even in the fully active gene in mature oligodendrocytes, indicating that they may be essential in maintaining proper chromatin structure.