Hybridization of a 3H-labeled bovine glucagon cDNA plasmid to human metaphase chromosomes revealed significant labeling of the distal portion of the long arm of chromosome 2. A large portion (37%) of the cells analyzed exhibited labeling of the 2. A significant percentage (40%) of the labeled sites on the 2 were in segment 2q36----37. Therefore, the human glucagon gene (GCG), was assigned to this segment. Localization of the glucagon gene, whose chromosomal assignment was previously not known, demonstrates the general applicability of in situ hybridization as a powerful gene mapping technique.