This article describes the development of micro-opioid receptor (MOR) binding and GTPgammaS functional SPAs as improved screening tools for the identification of MOR antagonists. Opioid receptors are members of the seven-transmembrane G protein-coupled receptor (GPCR) family and are involved in the control of various aspects of human physiology, including pain, stress, reward, addiction, respiration, gastric motility, and pituitary hormone secretion. Activation of the MOR initiates intracellular signaling pathways leading to a reduction in intracellular cyclic AMP levels, inhibition of calcium channels, and activation of potassium channels resulting in a reduction of the excitability of neurons. Characterization of opioid receptor ligand binding has traditionally been accomplished through the use of low throughput filtration-based binding assays, whereas functional activity has been based upon cyclic AMP measurements or filtration-based GTPgammaS functional assays. This report describes the development of a MOR displacement binding SPA using the radiolabeled antagonist [(3)H]diprenorphine ((3)H-DPN). The assay was optimized using statistical experimental design and demonstrates the stability and robustness necessary for HTS. The assay was biased toward the identification of MOR antagonists through the addition of Na(+). Our assay conditions also minimized the phenomenon of ligand depletion, a problem commonly observed in low-volume assays using high receptor-expressing cell lines. The optimized procedure revealed (3)H-DPN affinity constants at the MOR that were consistent with results obtained using filtration methods (K(D) (SPA) = 1.89 +/- 0.24 nM, K(D) (filtration) = 1.88 +/- 0.35 nM). The binding SPA identified known opioid receptor modulators contained within the Library of Pharmacological Active Compounds (LOPAC) cassette, and the GTPgammaS scintillation proximity assay (SPA) was used to confirm the functional activity of the LOPAC antagonists acting at the MOR. Conversion of the ligand binding and GTPgammaS functional assays to a homogeneous SPA generated a simple assay with dramatically increased throughput. Data from the development and implementation of the displacement binding and GTPgammaS functional SPAs are presented.