Treatment of patients with CCK2 receptor antagonists potentiates pain relief induced by mu-opioid (MOP) agonists. In attempt to enhance this effect with a single bivalent ligand, we connected pharmacophores of non-peptidyl CCK2 receptor antagonist and MOP receptor agonist with a spacer. Spacer length of 16-21 atoms was consistent with simultaneous binding to both receptors, however provided no advantage in biological activity from that of two individual ligands (J Med Chem, 2009). Here, we extend this to examine effects of ligand tethering on receptor regulation. We prepared a series of CHO cell lines stably expressing yellow fluorescent protein (YFP)-tagged single receptor constructs or both of these receptors tagged with half of YFP (YN attached to one receptor and YC attached to the other). The YFP halves were not fluorescent until brought into spatial approximation to reconstitute YFP. These receptors bound their specific ligands effectively. The MOP agonist signaled normally and resulted in MOP receptor internalization. The CCK2 receptor antagonist did not stimulate receptor internalization. In the dual receptor-expressing cell line, bivalent ligands capable of binding both receptors simultaneously effected YFP fluorescence at the cell surface, and this signal internalized in a time- and temperature-dependent manner. Bivalent ligands with spacer arms too short to occupy both receptors simultaneously did not result in such a signal. Thus, a bivalent ligand is able to stimulate the association of two non-spontaneously-dimerizing receptors on the cell surface, and both of these receptors are internalized in response to binding a ligand of one receptor that stimulates internalization. Tethering provides a mechanism for dragging other surface molecules into the endocytic pathway.