In pursuit of truncated glucagon analogues that can interact with the glucagon receptor with substantial binding affinity, 23 truncated glucagon analogues have been designed and synthesized. These truncated analogues consist of several fragments of glucagon with 11 or 12 amino acid residues (1-4), conformationally constrained analogues containing the sequence of the middle region of glucagon (5-15), and truncated analogues containing the sequence of the C-terminal region (16-23). Biological assays of these analogues showed that the truncated glucagon analogues with the sequence of the C-terminal region possess significantly better binding affinity compared to the truncated analogues with the sequence of the middle region, and these analogues (17-23) demonstrated potent antagonistic activity (pA(2) values between 6.5 and 7.5). On the basis of these results, it can be suggested that glucagon interacts with its receptor with two hydrophobic patches located in the middle and the C-terminal regions of glucagon, and both hydrophobic patches are necessary for significant receptor recognition. These two hydrophobic binding motifs, located in two different regions of glucagon, appear to be the reason why the earlier attempts to obtain truncated analogues with good binding affinity did not result in any success. Long peptide hormones such as glucagon seem to require more than one binding pocket on the receptors for maximal interaction.