In order to determine the approximate molecular size of the plasma glucagon immunoreactivity originating in the pancreas and glucagon-like immunoreactivity (GLI) derived from the gastrointestinal tract, extracts of plasma, obtained from dogs under various conditions, were subjected to P-10 Bio-Gel filtration and the elution volume of the peaks of immunoreactivity was compared with that of insulin-I-131 and glucagon-I-131 markers. In an assay system which measures both gastrointestinal GLI and pancreatic glucagon, an extract of fasting plasma was found to have two peaks of immunoreactivity, one peak in the glucagon-I-131 zone, the other in the preinsulin- I-131 zone. These peaks could not be discerned in an assay system specific for pancreatic glucagon and must, therefore, be of gastrointestinal origin. Plasma obtained from the mesenteric vein of a partially depancreatized dog following an intraduodenal glucose load contained the same two peaks which again reacted poorly in the glucagonspecific assay system, indicating gastrointestinal origin. Pancreaticoduodenal vein plasma obtained from a dog during amino acid infusion revealed a very large peak of immunoreactivity in the glucagon-I-131 zone and a very small peak in the preinsulin-I-131 zone. Both peaks were readily measured in the glucagon-specific assay system, indicating their pancreatic origin. The eluates of the glucagon-I-131-sized peak had glucagon-like glycogenolytic activity in the perfused rat liver system and appeared to be identical to pancreatic glucagon. It is concluded that the pancreatic glucagon and gastrointestinal glucagon-like immunoreactivity extractable from plasma have the same molecular size in their tissues of origin.