We investigated the effects of increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP) on genes associated with oligodendrocyte differentiation in an immortalized glial cell line, 6E12, derived from the spinal cord of an MBP-SV40 large T-antigen transgenic mouse. Raising intracellular levels of cAMP induced expression of oligodendrocyte differentiation antigens recognized by O4 and anti-galactocerebroside antibodies, up-regulated expression of the proteolipid protein (PLP) gene, and down-regulated glial fibrillary acidic protein (GFAP) expression. There was no treatment effect on myelin-associated glycoprotein (MAG) expression. These phenotypic changes are consistent with oligodendrocyte differentiation. Treatment of 6E12 cells with dibutyryl cyclic AMP (DBC) down-regulated myelin basic protein (MBP) gene expression, perhaps, because it also up-regulated expression of a putative MBP repressor SCIP/Tst-1. Moreover, the 6E12 cells expressed high levels of MBP mRNA but no MBP translation products were detected in the presence or absence of DBC. This immortalized glial cell line is proposed as a CNS model for cAMP-modulated myelin gene expression and for post-transcriptional regulation of MBP.