Chromaffin cells of the adrenal medulla are neural crest-derived neuroendocrine cells that express neuropeptide genes in vivo and in vitro. As such these cells are useful for examining tissue- and cell-specific regulation of the enkephalin gene. We previously demonstrated that the chromatin configuration of the enkephalin gene correlated with its tissue-specific expression in the adrenal medulla and primary chromaffin cell cultures. In this study we examine and characterize binding of transcription factors to the enkephalin promoter/enhancer region. Gel shift analyses of this region with extracts from chromaffin cells and PC12 cells (a pheochromocytoma cell line that does not express the enkephalin gene) demonstrate that all detectable binding is to ENKCRE-2, a cyclic AMP response-like element, and that the binding is cell specific. Gel shift and supershift analyses show that, unlike reports demonstrating that binding activity in the CNS is composed of the cyclic AMP response element binding protein, CREB, the majority of protein binding in chromaffin cells is from the AP-1 family of transcription factors. This binding is composed of c-Jun, JunD, and possibly a novel Fos-related protein(s). These data suggest enkephalin gene expression in the adrenal gland is controlled by cell-specific binding of transcription factors from the Fos/Jun families to the enkephalin CRE-2 element. Furthermore, these data suggest at least two different modes of enkephalin gene regulation exist between endocrine and neuronal tissues.