Glucagon, the second major glucose-regulated hormone in the control of glucose homeostasis, functions as a counter-regulator to insulin and is specifically produced by the pancreatic alpha cells. Its excessive biosynthesis and secretion is associated with diabetes mellitus. The expression of the proglucagon gene has been demonstrated to be regulated by a cAMP-dependent pathway through cAMP-response element-binding protein (CREB) and possibly other transcription factors bound to its cAMP-response element (CRE)/activated transcription factor (ATF) site. Elsewhere we have shown that ATF3, a member of the ATF/CREB subfamily of the basic leucine zipper domain proteins, is expressed predominantly in the alpha cells of the pancreatic islets. In our attempts to further dissect the role of ATF3 proteins in alpha cells, we have identified and characterized a novel alternatively spliced form, ATF3b, and have compared the specific binding ability of ATF3 and ATF3b on the CRE/ATF motif of the proglucagon promoter. Our findings indicate the existence of a novel mechanism by which the transcription of the proglucagon gene is regulated in response to cAMP signals, in addition to CREB and in relation to glucose fluctuations in pancreatic alpha cells.