The glucagon gene is specifically expressed in the alpha cells of pancreatic islets. The promoter of the glucagon gene is responsible for this specificity. Within the promoter, the upstream promoter element G1 is critical to restrict expression to the alpha cells. We define here a composite DNA control element, G4, localized upstream of G1 between nucleotides -100 and -140 which functions as an islet-specific activator in both glucagon- and insulin-producing cells but not in nonislet cells. G4 contains at least three protein binding sites. The most proximal site, E2, is highly homologous to the E1, SMS-UE, and B elements of the rat insulin I, somastatin, and elastase I genes, respectively, and interacts with a pancreas-specific complex; the distal site, E3, represents an E box which is identical to the E boxes of the rat insulin I and II genes and binds to a complex similar or identical to IEF1 which has been implicated in the tissue-specific control of insulin gene expression. These two sites necessitate a third element, the intervening sequence, to activate transcription. We conclude that the first 140 bp of the glucagon gene promoter contains at least two DNA control elements responsible for pancreatic alpha-cell-specific expression: G4, an islet cell-specific element sharing common binding sites with the insulin gene, and G1, which restricts glucagon gene expression to the alpha cells. This double control of specificity might have relevance during islet cell differentiation.