Previous studies have demonstrated that glucagon-superfamily peptides stimulate insulin release from the pancreatic islets in a glucose dependent manner. In this study we have carried out a structure-activity study of their insulinotropic activity using a rat pancreas perfusion with 5.5 mM glucose concentration. The following peptides were examined: glucagon-like peptide-1(7-36)amide (tGLP-1), glucagon, gastric inhibitory peptide (GIP), peptide having an amino-terminal histidine and carboxy-terminal isoleucine amide (PHI), vasoactive intestinal polypeptide (VIP), growth hormone releasing factor(1-29)amide (GRF), GRF(1-27)amide and synthetic hybrid-peptides of PHI-GRF, PHI(1-11)-GRF(12-27) and PHI(1-20)-GRF(21-27). Their potencies were evaluated as: tGLP-1 = GIP > glucagon > PHI = VIP > PHI(1-20)-GRF(21-27) > PHI(1-11)-GRF(12-27) >> GRF(1-29) = GRF(1-27). It is clear that 0.1 nM tGLP-1 stimulated insulin release, whereas 1 microM GRF(1-29) did not. These results indicate that 1) in addition to N-terminal amino acid (histidine or tyrosine), position 4 (glycine), position 9 (aspartic acid) and position 11 (serine) in the amino acid sequence are important for their insulinotropic activity, 2) not only the N-terminal portion but also the C-terminal portion of these peptides contribute to their insulinotropic activity.