Opioids have been the mainstays to alleviate pain for millennia. The opioid receptors responsible for analgesia are G protein-coupled receptors (GPCRs) which bind several endogenous ligands and couple to multiple isoforms of the G_i/o family of G proteins. Numerous studies have promoted a theory of requisite opioid receptor oligomerization. However, recent reports have illustrated that prototypical GPCRs are fully capable of activating their cognate G proteins as monomers. We therefore set out to determine if the monomeric form of the 1A-opioid receptor (MOR) is the minimal functional unit for efficient G protein coupling and signaling. To investigate this question a yellow fluorescent protein-MOR fusion protein (YMOR) was constructed and expressed in insect cells to facilitate high expression levels. YMOR displays appropriate function, as it binds ligands with high affinity, induces agonist-stimulated [ ³⁵S]GTPγS binding to Gα_i, and is allosterically regulated by coupled G_i protein heterotrimer in insect cell membranes. A novel purification scheme was developed to purify active YMOR, which was then reconstituted into a phospholipid bilayer in the form of high density lipoprotein (HDL) particles. Single particle imaging of fluorescently-labeled YMOR confirmed the receptor was monomeric in HDL particles. Just as in membrane preparations, monomeric YMOR binds alkaloid and peptide antagonists and agonists with high affinity, activates coupled G_i and G_o heterotrimers, and is subject to allosteric regulation by both G _i and G_o. Furthermore, experiments measuring Gα stimulation suggest that monomeric YMOR can support differential activation of G _i protein heterotrimers. These data illustrate that the monomeric form of MOR is indeed the minimal functional unit for G protein coupling. In additional studies, single molecule imaging of a Cy3-labeled MOR agonist, [Lys ⁷, Cys⁸]dermorphin, illustrate a novel method for investigating GPCR-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. While the role of oligomerization of opioid receptors continues to be debated, this work has shown that it is not essential for G protein coupling in vivo. It is possible that opioid receptor oligomerization is involved in differential arrestin recruitment and/or desensitization and internalization, where it may play a role in the development of tolerance to opioids in vivo.