The authors report the characterization of a novel cyclic adenosine monophosphate (cAMP)-responsive luciferase (Luc) reporter that exhibits optimal performance in high-throughput screens of agonist binding at G protein-coupled receptors (GPCRs). This reporter (RIP1-CRE-Luc) incorporates a nonpalindromic cAMP response element (CRE) originally identified within the 5' promoter of the rat insulin 1 gene (RIP1). When multimerized and fused to the coding sequence of firefly luciferase, the CRE of RIP1 allows for the efficient activation of luciferase expression by cAMP-elevating agents or by cAMP itself. Of primary importance is the demonstration that RIP1-CRE-Luc does not exhibit the relatively high levels of basal luciferase activity inherent to reporters incorporating the palindromic CRE first identified in the somatostatin gene promoter. Furthermore, studies of HEK cells expressing class II GPCRs for the cAMP-elevating hormones GLP-1, GIP, and glucagon demonstrate that RIP1-CRE-Luc affords a much wider dynamic range of activation upon exposure to agonist. Such properties of RIP1-CRE-Luc indicate its usefulness as a new and powerful tool for the identification of small-molecule compounds with receptor-stimulating actions or for the identification of constitutively active orphan receptors with cAMP-signaling properties.