Glucagon is a 29-amino acid polypeptide hormone synthesized by the A cells of the endocrine pancreas. 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 (Mr) approximately 18,000 which is five to six times larger than glucagon. Glucagon-containing polypeptides and immunoreactants of various sizes have also been described from stomach, intestine, brain and salivary gland. 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.