M*easurements of plasma concentrations of the incretin hormone GLP-1 are complex because of extensive molecular heterogeneity. This is partly due to a varying and incompletely known degree of carboxyterminal amidation. Given that virtually all GLP-1 assays rely on a C-terminal antibody, it is essential to know whether or not the molecule one wants to measure is amidated. We performed a detailed analysis of extractable GLP-1 from duodenum, proximal jejunum, distal ileum, caecum, proximal colon and distal colon of mice (n = 9), rats (n = 9) and pigs (n = 8) and determined the degree of amidation and whether this varied with the six different locations. We also analyzed the amidation in 3 GLP-1 secreting cell lines (GLUTag, NCI-H716 and STC-1). To our surprise there were marked differences between the 3 species with respect to the concentration of GLP-1 in gut. In the mouse concentrations increased continuously along the intestine all the way to the rectum, but were highest in the distal ileum and proximal colon of the rat. In the pig very little or no GLP-1 was present before the distal ileum with similar levels from ileum to distal colon. In the mouse GLP-1 was extensively amidated at all sampling sites, whereas rats and pigs on average had around 2.5 and 4 times higher levels of amidated compared to non-amidated GLP-1, although the ratio varied depending of the location. GLUTag, NCI-H716 and STC-1 cells all exhibited partial amidation with 2-4 times higher levels of amidated compared to non-amidated GLP-1.