AbstractMu and delta opioid receptors (MORs and DORs) were co‐expressed as fusion proteins between a receptor and a pertussis insensitive mutant Gαi/oprotein in human embryonic kidney 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Gαi/oproteins by pertussis toxin. Co‐expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonistd‐Phe‐Cys‐Tyr‐d‐Trp‐Arg‐Thr‐Pen‐Thr‐NH2. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic‐deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonistN,N(CH3)2‐Dmt‐Tic‐NH2. When a MOR fused to a non‐functional Gα subunit was co‐expressed with the DOR‐Gα protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced DOR coupling to its tethered Gα subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the DOR and also results in activation of the Gα subunit fused the DOR.