We have recently demonstrated that mRNA expression of cyclic AMP (cAMP) response element-binding protein (CREB) is down-regulated in CATH.a cells (a neural-derived cell line) by activation of the cAMP pathway. We now demonstrate that this down-regulation can be accounted for by a decrease in the rate of CREB gene transcription. It was found that cycloheximide, a protein synthesis inhibitor, prevented the forskolin-induced decrease in CREB mRNA levels in CATH.a cells. Nuclear run-on assays demonstrated that forskolin decreased the rate of CREB transcription by close to 50%. Moreover, forskolin decreased chloramphenicol acetyltransferase (CAT) activity in CATH.a cells transiently transfected with a construct containing 1,240 bp of CREB promoter fused to a CAT reporter plasmid. Possible mechanisms by which activation of the cAMP pathway leads to a decrease in CREB gene transcription are discussed.