Antibody titers in rabbits immunized with glucagon conjugated to albumin using difluorodinitrobenzene rose rapidly. Under conditions of immunoassay, less than 2 nl of serum from 2 of 4 animals and approximately 4 nl from the other two was required to bind 50% of 10 pg of [125I]iodoglucagon 100 days after immunization. The dissociation constants of the 2 higher titer antisera for glucagon were approximately 1 .times. 10-10 M, and their binding capacities for the hormone, about 50 .mu.g/ml. Competitive binding assays showed that neither of these antisera cross-reacts with the glucagon-like, immunoreactive peptides extracted from intestine to greater than 2.5%. In contrast, hens produced antisera which were reactive with the intestinal material and which bound only 0.3 .mu.g of glucagon per ml. There were no consistent differences in the abilities of specific and nonspecific antisera to react with selected fragments of pancreatic glucagon.