Studies were undertaken to investigate the regulation of myelin-specific mRNA expression in cultured cells. Three experimental systems were investigated: primary oligodendrocytes grown as enriched cell populations, primary oligodendrocytes grown in the presence of chick spinal cord neurons, and C6 cells. cDNA probes specific for the myelin proteolipid mRNA and the myelin basic protein mRNA were used to quantitate proteolipid and myelin basic protein mRNA levels in cells under different experimental conditions. C6 cells expressed less than 0.2% of the proteolipid mRNA that was expressed in primary oligodendrocytes. Primary oligodendrocytes expressed the myelin-specific mRNAs for at least 104 days in culture, and the level of these mRNAs in cultures was elevated fourfold by coculturing rat oligodendrocytes with chick spinal cord neurons.