The level of proenkephalin mRNA in bovine adrenal chromaffin cells was studied in the presence of cycloheximide, an inhibitor of translation, and two modulators of proenkephalin synthesis, nicotine and 12-O-tetradecanoylphorbol-13-acetate (TPA). Cycloheximide (CHX) abolished the induction of proenkephalin mRNA expression and protein synthesis by these two modulators, indicating that de novo protein synthesis was necessary for proenkephalin gene activation. The transcriptional regulatory regions of the proenkephalin gene displayed an extremely high degree of interspecies sequence conservation between humans, rats, and cows. In addition, molecular analyses of the human proenkephalin gene defined a cluster of responsive elements, designated as ENKCRE-1, ENKCRE-2, and AP-2; ENKCRE-2 acted functionally like both an AP-1 motif and a CAMP responsive element (CRE). When oligonucleotides containing ENKCRE-1, ENKCRE-2, AP-2, AP-1, and CRE motifs were used in protein-DNA gel mobility retardation experiments, the induction of ENKCRE-2/AP-1 activity correlated well with the level of proenkephalin mRNA induction. This ENKCRE-2/AP-1 complex could be inhibited by a specific c-Jun antiserum and was super-shifted by a polyclonal antibody against the Fos family of proteins. Furthermore, Western blot analysis suggested that c-Jun and Fos-related antigens rather than c-Fos per se were components of an ENKCRE-2/AP-1 complex. Thus, Fos-related proteins apparently form a complex with c-Jun that transactivates the proenkephalin gene.