The aim of this study was to explore individual amino acid-stimulated GLP-1 responses and the underlying stimulatory mechanisms, as well as to identify the amino acid-sensing-receptors involved in amino acid-stimulated GLP-1 release. Experiments were primarily based on isolated perfused rat small intestines, which have intact epithelial polarization allowing discrimination between luminal and basolateral mechanisms as well as quantitative studies of intestinal absorption and hormone secretion. Expression analysis of amino acid sensors on isolated murine GLP-1 secreting L-cells was assessed by qPCR. We found that L-valine powerfully stimulated GLP-1 secretion but only from the luminal side (2.9-fold increase). When administered from the vascular side, L-arginine and the aromatic amino acids stimulated GLP-1 secretion equally (2.6-2.9 fold increases). Expression analysis revealed that Casr expression was enriched in murine GLP-1 secreting L-cells, whereas Gpr35, Gprc6a, Gpr142, Gpr93 (Lpar5) and the umami taste receptor subunits Tas1r3 and Tas1r1 were not. Consistently, activation of GPR35, GPR93, GPR142 and the umami taste receptor with specific agonists or allosteric modulators did not increase GLP-1 secretion (P>0.05 for all experiments), whereas vascular inhibition of CaSR reduced GLP-1 secretion in response to luminal infusion of mixed amino acids. In conclusion, amino acids differ in their capacity to stimulate GLP-1 secretion. Some amino acids stimulated secretion only from the intestinal lumen, while other amino acids exclusively stimulated secretion from the vascular side, indicating that amino acid-stimulated GLP-1 secretion involves both apical and basolateral (post-absorptive) sensing mechanisms. Sensing of absorbed amino acids involves CaSR activation as vascular inhibition of CaSR markedly diminished amino acid stimulated GLP-1 release.