This study examined the signal transduction correlates ofmu opioid agonist efficacy in two systems:mu receptor-transfected mMOR-CHO cell and rat thalamic membranes. The potency and maximal stimulation of [³⁵S]GTPγS binding by various agonists was measured in the presence of excess GDP and compared with receptor binding affinity under identical assay conditions. Results showed that the relative maximal stimulation produced by these agonists was greater in mMOR-CHO cell than in rat thalamic membranes; some drugs that were full agonists in mMOR-CHO cells were partial agonists in the thalamus, and some partial agonists in the transfected cells were full antagonists in the thalamus. Furthermore, there was receptor reserve for G-protein activation by some agonists in mMOR-CHO cell membranes, but no receptor reserve was detected in rat thalamic membranes. Saturation analysis of agonist-stimulated [³⁵S]GTPγS binding revealed that full agonists produced both a higher B _maxand apparent affinity of [³⁵S]GTPγS binding than partial agonists. Correlation of the B _maxand K _D of agonist-stimulated [³⁵S]GTPγS binding with agonist intrinsic efficacy revealed only a moderate correlation with either parameter alone, but a highly significant correlation (r > 0.9) with a combination of the two parameters (B _max/K _D). These results suggest that the intrinsic efficacy of agonists at G-protein-coupled receptors is determined primarily by the ability of the agonist-occupied receptor to promote high-affinity GTP binding to the G-protein and to catalytically activate a maximal number G-proteins.