Background and Purpose: Various GPCRs have been described as being modulated in a voltage-dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling. Experimental Approach: We investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer-based assays. Depolarizationinduced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials. Key Results: Studying μ receptor function and signalling in cells, we discovered that morphine-induced signalling was strongly dependent on the membrane potential (V M). This became apparent at the level of G-protein activation, G-protein coupled inwardly rectifying potassium channel (K ir 3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine-induced μ receptor activation was also observed at the level of K ir 3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met-enkephalin) or only moderately ([D-Ala 2 , N-Me-Phe 4 , Gly 5-ol]-enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors. Conclusion and Implications: Our results indicate a strong ligand-dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.