Structure—function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser2–Asp13)GIP, containing a negatively charged Asp residue in place of an Ala in position 13, was synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser2–Asp13)GIP to be 2.3 and >4 h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser2–Asp13)GIP (10−12to 10−7mol/l) was significantly less potent (60–90%; P<0.05 to P<0.001) than native GIP. The peptide failed to display antagonistic properties as it did not significantly alter insulin secretion when incubated in the presence of GIP (10−7 mol/l). These results demonstrate that despite increased resistance to DPP IV, substituting Ala in position 13 with a negatively charged Asp, thus producing the di-substituted analogue (Ser2–Asp13)GIP, significantly reduces biological activity, most likely due to modifications within the secondary structure.