Viable pancreatic islets were isolated from the pancreas of humans using modifications of the collagenase digestion and Ficoll gradient techniques. Gel filtration of tissue extracts following islet incubation in the presence of 3H tryptophan indicated that radioactivity becomes incorporated into at least two islet proteins. The larger of the two (LGI) has the approximate molecular size of proinsulin and the smaller coelutes with glucagon as determined by column standardization. Radioimmunoassay of the gel filtration eluate for glucagon revealed that both molecules have glucagon immunoreactivity. Gel filtration in the presence of 8M urea did not alter the elution pattern of the LGI molecule. Polyacrylamide gel electrophoresis was performed on these glucagon immunoreactive molecules. The 3H radioactivity and the glucagon immunoreactivity of the smaller molecule were found to co migrate electrophoretically with crystalline porcine glucagon and monodesamidoglucagon. With electrophoresis at high pH the electrophoretic mobility of the LGI molecule proved to be lower than that of glucagon. Partial tryptic degradation of the human LGI molecule yields 3H tryptophan labeled products having charge and immunologic characteristics indistinguishable from porcine glucagon and monodesamidoglucagon. Although further investigation is indicated, these results suggest that the human LGI molecule contains covalently bound glucagon and may thus serve as a precursor or an intermediate in human glucagon biosynthesis.