We are developing a system to control G protein signaling in vivo to regulate a broad range of physiologic responses. Our system utilizes G protein-coupled peptide receptors engineered to respond exclusively to synthetic small molecule ligands and not to their natural ligand(s). These engineered receptors are designated RASSLs (receptor activated solely by a synthetic ligand). We have made two prototype RASSLs that are based on the human κ opioid receptor. Small molecule drugs that activate the κ receptor are nonaddictive and safe to administer in vivo. Binding and signaling assays reveal 200-2000-fold reductions in the ability of our RASSLs to bind or be activated by dynorphin, an endogenous peptide ligand of the κ opioid receptor. In a high-throughput signaling assay, these prototype RASSLs expressed in Chinese hamster ovary K1 cells showed little or no response to a panel of 21 opioid peptides but still signaled normally in response to small molecule drugs such as spiradoline. Activation of a RASSL by spiradoline also caused proliferation of rat-1a tissue culture cells. These data provide evidence that G protein-coupled receptors can be made into RASSLs. The potential in vivo applications for RASSLs include the positive enrichment of transfected cells and the development of new animal models of disease.