We have shown previously that in oligodendrocytes, the transcription factor cyclic AMP response element binding protein (CREB) is maximally expressed immediately prior to the most rapid period of myelination in rat brain. We have begun to investigate the role of this protein during myelination by downregulating CREB synthesis in cultured oligodendrocytes using an antisense deoxyoligonucleotide directed against CREB mRNA. Neonatal oligodendrocytes were grown for 4 days in a chemically defined medium (CDM) after which intracellular delivery of CREB antisense oligonucleotide was facilitated by using a liposome preparation. Control cultures were treated in a similar manner but in the presence of CREB sense oligomer. Immediately after transfection, cells were cultured for 3 days in CDM in the presence or absence of the cyclic AMP (cAMP) analogue N6, O21-dibutyryl cAMP (db-cAMP). In these cultures, myelin basic protein (MBP) expression was investigated by immunocytochemistry and Western blot analysis. Treatment of control cultures with db-cAMP resulted in a significant increase in the number of MBP positive cells which was abolished when the cells were treated with CREB antisense oligonucleotide. MBP positive cells in control cultures treated with db-cAMP have extended and highly branched MBP positive processes. In contrast, MBP positive cells in either control cultures grown in the absence of db-cAMP or cultures grown in the presence of db-cAMP but treated with CREB antisense oligonucleotide showed shorter and less complex processes and the MBP immunoreactivity appeared to be concentrated in the cell body. These observations suggest that CREB is at least one of the mediators in the induction of oligodendrocyte differentiation by cAMP.