Glucagon is a 29-amino acid polypeptide hormone synthesized by the A cells of the endocrine pancreas^1–4. Its primary site of action is the liver where it stimulates glycogenolysis, gluconeogenesis and ketogenesis. In mammals, biosynthetic studies have shown that glucagon is derived from a precursor of molecular weight (M_r) approximately 18,000 which is five to six times larger than glucagon⁵. Glucagon-containing polypeptides and immunoreactants of various sizes have also been described from stomach, intestine, brain and salivary gland³. Here, we have determined the structure of hamster pancreatic preproglucagon from the sequence of its cDNA. This 180-amino acid precursor contains the sequence of glucagon and two glucagon-like polypeptides arranged in tandem. The precursor also contains the sequences of several non-pancreatic glucagon-containing polypeptides which suggests that, in mammals, both pancreatic and non-pancreatic glucagon and glucagon-containing polypeptides may be derived from a common precursor by tissue-specific processing. We have tentatively identified each of the glucagon-like immunoreactants which have been described with respect to the sequence of proglucagon and have proposed a scheme for the processing of pancreatic proglucagon.