Receptor:effector coupling is central to the function of G protein-coupled receptors. Ligand dependent differences in receptor:effector interactions underlie the well-characterized phenomenon of differential efficacy and are presumed to underlie biased signaling. We have previously demonstrated that the divergent efficacy of two calcitonin receptor (CTR) ligands (salmon & human calcitonin) is a consequence of differences in nucleotide handling at the Gαs effector1, rather than the absolute receptor:effector affinity. In two different protein-population biophysical assays the average conformation of Gαs bound to the CTR differs according to the bound ligand. We observe a ligand-dependent difference in apparent GTP affinity and GTP on-rate that are correlated with the conformation of the receptor-bound Gαs. We interpret these disparities as ligand-dependent receptor conformational differences being communicated to the effector, resulting in ligand-dependent Gαs conformations. The variation in nucleotide handling could be due to an absolute difference in Gαs conformation or alternatively due to divergent exchange rates between Gαs-effector conformations. In the former case, this would point to distinct absolute conformations being important for determining efficacy, whereas the latter would suggest that receptor:effector dynamics may be more important in determining efficacy. To refine our model we have developed a novel bi-orthogonal bi-arsenical labeling approach to label the G protein hetero-trimer with a Cy3 / Cy5 FRET (Förster resonance energy transfer) pair. We have used this labeling strategy in single molecule FRET-FLIM (Fluorescence lifetime imaging) experiments to distinguish between ligand-dependent differences in either absolute or dynamic Gαs conformation as well as to understand the transitions between nucleotide free and bound Gαs conformations. We believe this data will inform approaches to designing agonists with particular efficacy profiles.