We developed antisera and radioimmunoassays against synthetic replicas of glucagon-like peptide-1 (1–36) and -2, predicted products of the glucagon precursor, and against glucagon-like peptide-1 (7–36) identical to the sequence of glucagon-like peptide-1, but lacking its first six N-terminal amino acids. With these tools, we studied the localisation and molecular nature of glucagon-like immunoreactivity in human pancreas, small intestine and plasma. By immunohistochemistry glucagon-like peptide-1, and glucagon-like peptide-2 immunoreactivity coexisted with glucagon in pancreatic islet cells and with enteroglucagon in small intestinal enteroglucagon-producing cells. By chromatography of tissue extracts we found that glucagon-like peptide-1 and glucagon-like peptide-2-immunoreactivities in the human pancreas (307±51 and 107±37 pmol/g tissue) were mainly contained in a large peptide, whereas in the small intestine glucagon-like peptide-1 and glucagon-like peptide-2 immunoreactivities were found in separate smaller molecules (49±21 and 77±28/g tissue). By isocratic high pressure liquid chromatography of the large pancreatic glucagon-like peptide we found that this peptide is heterogeneous. By chromatographic analysis glucagon-like peptide-1 immunoreactivity in fasting plasma was mainly found in a large peptide corresponding to the pancreatic form, while after a meal a smaller molecular form coeluting by gel filtration with glucagon-like peptide-1 predominated.