The transcription factor CREB [cyclic AMP response element (CRE)-binding protein] is activated by several kinase pathways on phosphorylation of serine-133. Phosphorylation of CREB at serine-133 is required for the induction of target gene expression. The proenkephalin gene is a target of cyclic AMP-dependent agonists like forskolin, and its expression is driven by the enhancer element CRE-2. It has been shown that CREB binds CRE-2 in extracts from striatum and hypothalamus. However, these studies did not show a functional requirement for CREB serine-133 phosphorylation in CRE-2 function. We demonstrate that CREB binds CRE-2 in primary astrocyte cultures and that transcriptional activation of CRE-2 requires CREB phosphorylation at serine-133. In addition, it has recently been shown that, at least in some contexts, CREB phosphorylation is not sufficient to activate target gene expression and that another intracellular signal seems to be required. Therefore, we also sought to determine if another signaling event, in addition to CREB phosphorylation, might be involved in cyclic AMP-mediated induction of the proenkephalin gene. We have found that the inhibition of src-related nonreceptor tyrosine kinases blocks forskolin-induced proenkephalin gene expression without having any effect on serine-133-phosphorylated CREB levels and that constitutively activated src kinase can activate the proenkephalin promoter.