Glucagon, a pancreatic peptide hormone of 29 amino acids that regulates carbohydrate, fat, and protein metabolism, is one of a family of structurally similar regulatory peptides which include GH-releasing hormone, vasoactive intestinal peptide, secretin, and gastric inhibitory peptide. The synthesis of glucagon involves its specific proteolytic cleavage from preproglucagon, a large polyprotein precursor. To facilitate analyses of the cellular processing of pre-proglucagon and to begin studies of the regulation of glucagon gene expression in the rat, we have cloned and sequenced two cDNAs derived from rat neonatal pancreas. The cDNAs represent close to the entire transcriptional sequence of the glucagon gene and encode a pre-proglucagon of 180 amino acids. The coding region of pre-proglucagon contains, in addition to the sequence of glucagon, the sequences of two peptide domains that are related in their structures to glucagon. Glucagon and the two glucagon-like peptides are flanked in the precursor by pairs of basic amino acids characteristic of the sites that are cleaved during the posttranslational processing of pro-hormones. Northern blot analyses indicate the presence of a single mRNA of 1400 +/- 100 nucleotides in the rat pancreas, and results of Southern blotting of rat liver genomic DNA are consistent with the existence of a single chromosomal gene. Comparisons of the nucleotide sequence of the rat pre-proglucagon cDNA with those of the two pre-proglucagons of the anglerfish pancreas encoded by two separate genes indicate that the pre-proglucagon genes probably evolved by intragenic duplications of a DNA segment corresponding to the coding sequences of glucagon and the glucagon-like peptides.