We have investigated the properties of derivatives of the 29-amino-acid polypeptide hormone, glucagon, which had varying numbers of amino acids cleaved from the carboxyl terminal portion of the molecule. These derivatives included a 27-amino-acid fragment made by cleavage of the native molecule with cyanogen bromide and a 23-amino-acid fragment made synthetically. We found both of these derivatives capable of stimulating the conversion of ATP to cyclic AMP in in vitro assays using rat liver homogenates. The concentration of these peptides, which were required to produce enhanced adenyl cyclase activity, was higher than that of glucagon, and in the case of the 1–23 derivative it was several orders of magnitude larger. The requirement for higher concentrations of peptide is expected, as removal of a large portion of the total number of amino acids will proportionately decrease the free energy of dissociation of the peptide from the membrane receptor, thus changing the equilibrium constant for binding by several orders of magnitude. Circular dichroism and ultra-centrifuge studies of these peptides, as well as a 21-amino-acid fragment made by cleavage of the native molecule with carboxypeptidase, indicated that the shorter glucagon analogues have structures similar to that of the native molecule although somewhat less ordered.