A cAMP response element (CRE) has been identified in the proximal 5'-flanking region of the rat glucagon gene, and activation of the cAMP-dependent pathway in fetal rat intestinal cells leads to an increase in the levels of glucagon mRNA transcripts. In contrast, the human glucagon gene does not contain a similar CRE, and the results of studies using immortalized rat and hamster islet cell lines have suggested that glucagon gene expression may not be regulated by cAMP. To reconcile these observations, we have studied the control of glucagon gene expression. Incubation of primary rat islet cell cultures with forskolin in the presence of low (0.5 g/liter) or high (2.0 g/L) glucose resulted in a 2- to 3-fold increase in the levels of glucagon mRNA transcripts. Forskolin also stimulated the secretion and synthesis of immunoreactive glucagon. The importance of the protein kinase-A-dependent pathway in the regulation of glucagon gene expression was also examined in hamster islet InR1-G9 cells. Cotransfection of a glucagon-chloramphenicol acetyltransferase (CAT) fusion gene containing the glucagon CRE and a cDNA encoding the catalytic subunit of protein kinase-A resulted in stimulation of glucagon-CAT activity in hamster islet cells. Catalytic subunit cotransfection also activated somatostatin-CAT, but no activation of RSVCAT was detected. The results of these experiments suggest that the rat glucagon gene is regulated by a protein kinase-A-dependent pathway in the endocrine pancreas.