We have previously shown that in cell extracts from rat striatum, cyclic AMP response element (CRE) binding protein (CREB), rather than AP-1 proteins, preferentially interacts with the CRE-2 element of the proenkephalin second messenger-inducible enhancer, even under conditions in which AP-1 proteins are highly induced. Here we use primary striatal cultures to permit a more detailed analysis of CRE-2 function and protein binding in relevant neural cell types. By transfection we find that in primary striatal cultures, as in transformed cell lines, the CRE-1 and CRE-2 elements are required for significant induction by cyclic AMP. We report that cyclic AMP induction of the proenkephalin gene in striatal cultures is protein synthesis independent, excluding a role for newly synthesized proteins like c-Fos. We also show that cyclic AMP induces CREB phosphorylation and that phosphorylated CREB interacts strongly with CRE-2 and weakly with CRE-1. The predominant protein bound to CRE-1 is not CREB, however, and remains to be identified. Despite some prior predictions, we do not find a role for c-Fos in cyclic AMP regulation of proenkephalin gene expression in neurons.