High-resolution analysis of ligand-mediated positive efficacy was obtained by measuring time-dependent Ca⁺⁺ responses at a mutant Thr³⁷⁰Lys α_2B AR under conditions of fusion or co-expression with either a chimeric G_αq/il protein or a promiscuous G_α15 protein. Clear differences in the magnitude of the Ca⁺⁺ response and/or kinetic properties were observed for the native ligand (-)-adrenaline by each of the Thr³⁷⁰Lys α_2B AR/G_α protein expression conditions. The putative antagonists atipamezole, dexefaroxan and idazoxan displayed significant positive efficacy at nanomolar concentrations regardless of the expression condition of the Thr³⁷⁰Lys α_2B AR, whereas RX 811059, the ethoxy-derivative of idazoxan, remained silent up to 10 μM. The type of G_α protein affected the magnitude of the Ca⁺⁺ response for the partial agonists only when the G_α protein was co-expressed, and not when the G_α protein was fused to the Thr³⁷⁰Lys α_2B AR. In conclusion, determination of Ca⁺⁺ responses by the mutant Thr³⁷⁰Lys α_2B AR constitutes a sensitive model to differentiate between antagonists that are commonly assumed to be similar and silent. This approach may reveal distinct molecular properties for α₂ AR ligands which appear difficult to differentiate at the wild-type receptor.