It is well established that glucagon is stored within the AZ -cells of the pancreatic islets, although up till now little is known about the cellular mechanisms involved in the biosynthesis of this hormone. The possibility that glucagon, like insulin, may be initially synthesized as a larger precursor has been considered on the basis of the recent demonstration of glucagonlike immunoreactive material of at least twice the molecular size of glucagon in pancreatic extracts of dog and several other species including man [ 11. In addition, recent reports have indicated the occurrence of a possible precursor molecule of glucagon in fish [2] and in pigeon islets [3], but there appears to date to be little information available about the mechanisms of glucagon biosynthesis and its regulation in mammalian islets. We report here investigations of the biosynthesis of glucagon in isolated guinea-pig islets of Langerhans during incubation in vitro for 17 hr or in tissue culture for up to 6 days. Tryptophan, labelled with tritium, was used as a marker for newly synthesized glucagon in the guinea-pig islets since this amino acid is present in the glucagon molecule [4] but not in the proinsulin [5] or insulin [6] molecule of any species studied so far. After incubation labelled proteins were extracted from the islets and analysed by gel filtration. At each time examined the extracts contained labelled proteins eluting in the void volume and in addition a protein of molecular weight about 18,000 which could not be dissociated by gel filtration in the presence of 8 M urea or 3 M acetic acid. The elution volume of this protein corresponded with that of a peak of glucagon-