Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and improves glycemic control in type 2 diabetes. In serum the peptide is degraded by dipeptidyl peptidase IV (DPP IV). The resulting short biological half-time limits the therapeutic use of GLP-1. Therefore, various GLP-1 analogues with alterations in cleavage positions were synthesized. GLP-1-receptor binding was investigated in RINm5F cells. Biological activity of the GLP-1 analogues was investigated in vitro by measuring cAMP production in RINm5F cells. GLP-1 analogues with modifications in position 2 were not cleaved by DPP IV and showed receptor affinity and in vitro biological activity comparable to native GLP-1. Analogues with alterations in positions 2 and 8, 2 and 9 or 8 and 9 showed a significant decrease in receptor affinity and biological activity. In vivo biological activity was tested in pigs. GLP-1 analogues were administered subcutaneously followed by an intravenous bolus injection of glucose. Plasma glucose and insulin were monitored over 4 h. Compared to native GLP-1, analogues with an altered position 2 showed similar or increased potency and biological half-time. Other GLP-1 analogues were less active. Despite the lack of degradation of these GLP-1 analogues by DPP IV in vitro, their biological action is as short as that of GLP-1, except for desamino-GLP-1, indicating that other degradation enzymes are important in vivo. Alterations of GLP-1 in positions 8 or 9 result in a loss of biological activity without extending biological half-time.