[Background] G-protein coupled receptors (GPCR) are known to form a dimerized homomeric or heteromeric receptors. However, coupling between dimerized receptor and β-arrestin is not well understood. We previously found that vasopressin can modulate morphine tolerance in ventral medulla through V1b receptor. However, how V1b, μopioid receptors and β-arrestin 2 are arranged is not known. Here, we employed three molecule BRET (bioluminescence resonance energy transfer), which occur between split luciferase and green fluorescent protein (GFP), to monitor association of a receptor-containing complex. [Method] V1b and μ opioid receptors were connected at their carboxyl-termini to each part of split luciferase. β-arrestin 2 was connected to an enhanced green fluorescent protein. HEK cell were transfected with genes for receptors and β-arrestin 2. After agonist stimulation, BRET signal was measured by a plate reader. [Results] Receptors connected with split luciferase were functional in term of generating their cellular responses. V1b and μopioid receptors formed homomeric or heteromeric receptor dimers, which were detect through luciferase intensities. Significant BRET signal was generated by interaction between receptor dimer and β-arrestin 2. [Conclusions] Our data suggested that the three-molecule BRET analysis can be applied to study interaction between receptor dimer and β-arrestin.