The glucagon-like peptide-1 receptor (GLP-1R) is a target for type 2 diabetes treatment. Due to the inconvenience of peptide therapeutics, small-molecule GLP-1R agonists have been studied. Compound 2 (6,7-dichloro-2-methylsulfonyl-2-N-tert-butylaminoquinoxaline) and compound B (4-(3-(benzyloxy)phenyl)-2-(ethylsulfinyl)-6-(trifluoromethyl)pyrimidine) have been described as small molecule, ago-allosteric modulators of GLP-1R. However, their modes of action at the GLP-1R have not been elucidated. Thus, in this study, we compared the mechanisms of action between these two compounds. When compound 2 was treated with endogenous or exogenous peptide agonists (GLP-1 and exenatide) or fragments of peptide agonists (GLP-1(9-36), Ex3, Ex4, and Ex5), the response curve of these peptide agonists shifted left without a change in maximum efficacy. In contrast, compound B potentiated the response and increased maximum efficacy. However, N-terminal truncated orthosteric antagonists including Ex7, Ex9, and Ex10, augmented the response of compound 2 at the GLP-1R but did not alter compound B activity. Intriguingly, when we co-treated compound 2 with compound B in CHO cells expressing full-length hGLP-1R or N-terminal extracellular domain-truncated GLP-1R, the activation of both types of receptors increased additively, implying that the N-terminus of the receptor is not involved in the modulation by compound agonists. We confirmed that these two compounds increased calcium influx by different patterns in CHO cells expressing GLP-1R. Taken together, our findings suggest that compounds 2 and B have different modes of action to activate GLP-1R. Further study to identify the putative binding sites will help in the discovery of orally available GLP-1R agonists. (C) 2011 Elsevier Inc. All rights reserved.