A clone containing the complete human glucagon gene was isolated and sequenced. The gene is approximately 9.4 kilobases in length and comprises six exons and five introns. The putative preproglucagon encoded by this gene, 180 amino acids in length and containing glucagon and two glucagon-like peptides, is very similar to that of other mammalian species (greater than 90% amino acid sequence homology). There is 88% nucleotide sequence homology between the proximal 130 base pairs of the 5' flanking regions of the human and rat glucagon genes. These sequences, highly conserved throughout evolution, are likely involved in the regulation of glucagon gene transcription.